Triazabutadienes as cleavable cross-linkers

ABSTRACT

Triazabutadiene molecules as cleavable cross-linkers adapted to cross-link components with click chemistry, e.g., clickable triazabutadienes. For example, in some embodiments, the triazabutadienes feature alkyne handles attached to the imidazole portion or the aryl portion of the triazabutadienes, wherein the alkyne handles can link to azide handles (e.g., azide handles disposed on other components) via click chemistry. Also described are methods of producing said clickable triazabutadienes and methods of use of said clickable triazabutadienes. The present invention also features methods of cleaving said clickable triazabutadienes, e.g., for liberating the diazonium species for further chemical reactions.

CROSS REFERENCE

This application is a continuation in part of U.S. patent applicationSer. No. 15/224,446 filed Jul. 29, 2016, which is a continuation in partof U.S. patent application Ser. No. 14/918,287 filed Oct. 20, 2015, nowU.S. Pat. No. 9,458,143, which is a continuation in part ofPCT/US15/35136 filed on Jun. 10, 2015, which claims priority to U.S.Provisional Application No. 62/010,861, filed Jun. 11, 2014, U.S.Provisional Application No. 62/109,170 filed Jan. 29, 2015, U.S.Provisional Application No. 62/114,735 filed Feb. 11, 2015, and U.S.Provisional Application No. 62/128,707 filed Mar. 5, 2015, thespecifications of which are incorporated herein in their entirety byreference.

This application is a continuation in part of U.S. patent applicationSer. No. 15/317,894 filed Dec. 9, 2016, which is a 371 application ofPCT/US15/35136 filed on Jun. 10, 2015, which claims priority to U.S.Provisional Application No. 62/010,861 filed Jun. 11, 2014, U.S.Provisional Application No. 62/109,170 filed Jan. 29, 2015, U.S.Provisional Application No. 62/114,735 filed Feb. 11, 2015, and U.S.Provisional Application No. 62/128,707 filed Mar. 5, 2015, thespecifications of which are incorporated herein in their entirety byreference.

BACKGROUND OF THE INVENTION

Triazabutadienes can be triggered to release a highly reactive diazoniumspecies in a pH-dependent way when placed in acidic conditions.Electron-rich phenyl systems such as resorcinol or tyrosine residues canreact with the diazonium compounds to form stable azobenzene products.Alterations of these triazabutadiene motifs allow for modification offunctionality, solubility, and other molecular properties. For example,triazabutadienes can be modified to function as cross-linkers; cleavageof the cross-linker triazabutadiene can liberate the diazonium species,in some cases near a site of interest.

The present invention features triazabutadienes as cleavablecross-linkers, wherein the triazabutadienes allow for cross-linking witha secondary component via click chemistry (copper (I) catalyzed azidealkyne cycloaddition), e.g., “clickable” triazabutadienes. In someembodiments, the clickable triazabutadienes comprise or are linked to afirst component (e.g., a protein, a drug, a surface, etc.) and via clickchemistry said first component can be cross-linked to a second component(e.g., another protein, surface, etc.).

The present invention also features methods of producing said clickabletriazabutadienes and methods of use of said clickable triazabutadienes.For example, the compositions of the present invention may be used asbiological cross-linkers and methods of the present invention may beused for biological methods such as detecting protein-proteininteractions, mapping drug-target interactions, discovering orcharacterizing host-pathogen interactions, etc. The present inventionalso features methods of cleaving said triazabutadienes, e.g., cleavingthe clickable triazabutadienes that has undergone click chemistry and isin the cross-linking state. In some embodiments, cleavage of thecross-linking triazabutadiene liberates the diazonium species; thus, thepresent invention also features methods that feature diazonium reactionsfollowing cleavage of said linking triazabutadienes.

SUMMARY

The present invention features clickable triazabutadiene according to(a) Formula B (see FIG. 11A) wherein X¹ comprises a terminal alkynehandle; or (b) Formula C (see FIG. 11A) wherein either X¹ comprises aterminal alkyne, X² comprises a terminal alkyne handle, or both X¹ andX² comprise a terminal alkyne handle; wherein A=S, O, or N; D=H,—CH═CH—CH=E-, halides, cyano, sulfonates, alkyl chain, ortrifluoromethyl; E=H, —CH═CH—CH=D-, halides, cyano, sulfonates, alkylchain, or trifluoromethyl; and wherein Y¹ comprises a tri-substitutedaryl group; wherein the alkyne handles are adapted to cross-link to anazide handle of a linking component via click chemistry. In someembodiments, the triazabutadiene comprises is linked to a peptide, anoligonucleotide, or a drug. In some embodiments, the linking componentwith the azide handle comprises a peptide, an oligonucleotide, or adrug.

The present invention also feature a method of detecting an interactionbetween a first component and a second component, said method comprisingcleaving a triazabutadiene linked to the first component via a firsttriazole formed from click chemistry and to the second component via asecond triazole formed from click chemistry, wherein cleaving thetriazabutadiene liberates a diazonium species whereupon the diazoniumspecies reacts with an electron-rich phenyl system to form a detectablesignal, said detectable signal being indicative of interaction betweenthe first component and the second component. In some embodiments, thetriazabutadiene is according to Formula C, wherein both X¹ and X²comprised a terminal alkyne handle prior to formation of the firsttriazole and second triazole via click chemistry.

The present invention also features a method of linking a functionalgroup or component to a clickable triazabutadiene, said functional groupor component comprising an azide handle, said clickable triazabutadienebeing according to (a) Formula B (see FIG. 11A) wherein X¹ comprises aterminal alkyne handle; or (b) Formula C (see FIG. 11) wherein either X¹comprises a terminal alkyne, X² comprises a terminal alkyne handle, orboth X¹ and X² comprise a terminal alkyne handle; wherein A=S, O, or N;D=H, —CH═CH—CH=E-, halides, cyano, sulfonates, alkyl chain, ortrifluoromethyl; E=H, —CH═CH—CH=D-, halides, cyano, sulfonates, alkylchain, or trifluoromethyl; and wherein Y¹ comprises a tri-substitutedaryl group; said method comprising subjecting the clickabletriazabutadiene and functional group or component to copper clickchemistry, wherein copper click chemistry links the clickabletriazabutadiene and functional group or component via formation of atriazole from the alkyne handle and the azide handle. In someembodiments, the functional group comprises a water solubilityfunctional group. In some embodiments, the component comprises apeptide, an oligonucleotide, or a drug.

Any feature or combination of features described herein are includedwithin the scope of the present invention provided that the featuresincluded in any such combination are not mutually inconsistent as willbe apparent from the context, this specification, and the knowledge ofone of ordinary skill in the art. Additional advantages and aspects ofthe present invention are apparent in the following detailed descriptionand claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows non-limiting examples of triazabutadiene molecules.

FIG. 2A shows triazabutadiene molecules undergoing decomposition todiazonium salts (and cyclic guanidine species). Note thereaction/equilibrium arrows are not to scale.

FIG. 2B shows a triazabutadiene molecule breaking down (in low pHconditions) to a diazonium species and a cyclic guanidine species.

FIG. 3 shows reductive cleavage of triazabutadiene molecules.

FIG. 4A shows light catalyzed cleavage of triazabutadiene molecules.

FIG. 4B shows photochemically generated bases. (i) A masked base maydecompose to reveal a basic nitrogen atom upon exposure to light; (ii)The basic nitrogen atom of a molecule obscured by a steric wall may bereversibly swung away in a photochemically triggered fashion; (iii) Theintrinsic basicity of a nitrogen-containing functional group may bealtered by a photochemical event.

FIG. 5A shows time-dependent photo-induced degradation oftriazabutadienes. (i) The reaction was monitored by comparing startingmaterials (A, C, D, and E) with product (B); (ii) Peak absorption andextinction coefficients for all of the compounds were excitable by theUV source used; (iii) Time-dependent conversion of compounds wasmeasured by NMR integration.

FIG. 5B shows (i) Compound A is rendered more basic upon exposure tolight; that basicity recovers (to some extent) in the absence of light;(ii) Oscillating UV irradiation provides a saw-tooth pH trend over time.

FIG. 5C shows the lone-pair of electrons on the N1 nitrogen atom becomesmore electron-rich upon isomerization from E to Z.

FIG. 5D shows the use of the photobase as a catalyst. (i) Structures ofwater-soluble Compound A versus organic soluble Compound F; (ii) TheHenry reaction between Compound G and Compound H was carried out at roomtemperature and varying amounts of catalyst; (iii) The reactions weremonitored by ReactIR™, following consumption of aldehyde Compound H.

FIG. 6A-FIG. 6H show non-limiting examples of triazabutadienes orreaction schemes involving triazabutadienes.

FIG. 7A, FIG. 7B, FIG. 7C, and FIG. 7D show non-limiting examples ofreaction schemes involving triazabutadienes.

FIG. 8 shows an example of a triazabutadiene molecule adapted to modifya protein.

FIG. 9 shows an example of a triazabutadiene molecule conjugated to anantibody, wherein the conjugate is used for labeling a protein ofinterest.

FIG. 10 shows an overview of Cu(II) and Cu(I) click chemistry.DIPEA=N,N-diisopropylethylamine.PMEDTA=1,1,4,7,7-pentamethyldiethylenetriamine.

FIG. 11A shows examples of structures of triazabutadienes adapted forclick chemistry (see Formula B and Formula C compared to Formula A).

FIG. 11B shows non-limiting examples of triazabutadienes adapted forclick chemistry, e.g., triazabutadienes comprising alkyne handles.

FIG. 12A shows synthesis of a triazabutadiene (TBD-6) comprising aterminal alkyne handle on the imidazole portion of the triazabutadiene.Note in some embodiments, n=1, n=2, n=3, n=4, etc.

FIG. 12B shows examples of azides for clickable triazabutadienesynthesis, e.g., synthesis of a triazabutadiene such as TBD-6 of FIG.12A. Note in azide 9′, PG stands for protecting group.

FIG. 12C shows synthesis of a triazabutadiene (TBD-7) comprising analkyne handle on the aryl portion of the triazabutadiene.

FIG. 12D shows click chemistry with a clickable triazabutadiene (TBD-6).Note in some embodiments, n=1, n=2, n=3, n=4, etc.

FIG. 12E shows click chemistry with a clickable triazabutadiene (TBD-7).

FIG. 13A shows synthesis of a triazabutadiene containing anN-hydroxysuccinimide ester.

FIG. 13B shows Compound 68.

FIG. 13C shows derivatives related to Compound 68.

FIG. 14A shows a proposed lysine reactive triazabutadiene that can berapidly modified in situ by click chemistry.

FIG. 14B shows preliminary synthesis of an alkyne containingtriazabutadiene Compound 75.

FIG. 14C shows proposed retrosynthesis of azide-containingtriazabutadiene Compound 76 from imidazolium Compound 77.

FIG. 14D shows benzylic azide Compound 78 can be made from previouslysynthesized alcohol Compound 79.

FIG. 15 shows lysine-reactive triazabutadiene Compound 80 is designed toself-immolate to return the starting lysine residue if the resultingdiazonium ion, Compound 81, fails to undergo a reaction with tyrosine.

FIG. 16A shows an example of cargo release from a triazabutadienemolecule.

FIG. 16B shows an example of how a prodrug is released.

FIG. 16C shows an example of a prodrug comprising a phenolic functionalgroup masked as a triazylidine moiety.

FIG. 16D shows an azide functional group reacted with a carbine toproduce an acid labile prodrug comprising a triazylidine moiety.

FIG. 17 shows an example of triazabutadiene molecules used as adhesives.

FIG. 18A shows an example of a triazabutadiene comprising an epoxide(Compound 1) that can be added to an epoxy resin (Compound B).

FIG. 18B shows an example of synthesis of a triazabutadiene (Compound 1)comprising an epoxide.

FIG. 18C shows an example of an alternative epoxide triazabutadiene.

FIG. 18D shows polystyrene (as if attached to the N₃ area of atriazabutadiene, e.g., in lieu of an epoxide as shown in Compound 1 ofFIG. 18A) and polyacrylamide (as if attached to the N₃ area of atriazabutadiene, e.g., in lieu of an epoxide as shown in Compound 1 ofFIG. 18A). Other classes of polymers may be contemplated in lieu ofepoxide, polyacrylamide, polystyrene, etc.

FIG. 19 shows synthesis of a fluorescent triazabutadiene.

FIG. 20A shows synthesis of a water-soluble triazabutadiene via clickchemistry. Note in some embodiments, n=1, n=2, n=3, n=4, etc.

FIG. 20B shows a synthetic scheme of a bis-triazole-triazabutadiene.Note in some embodiments, n=1, n=2, n=3, n=4, etc.

FIG. 21 shows a triazabutadiene comprising an epoxide (left) used toproduce a triazabutadiene with an alkyne group adapted for clickchemistry.

DETAILED DESCRIPTION OF THE INVENTION

I. Triazabutadiene Molecules

The present invention features triazabutadiene molecules. Non-limitingexamples of formulas for triazabutadiene molecules of the presentinvention are of shown in FIG. 1. For example, in some embodiments,triazabutadienes are according to Formula A. Examples of Formula A areshown as Formula I, II, III, and IV. The present invention is notlimited to Formula A, Formula I, Formula II, Formula III, and FormulaIV. Referring to FIG. 1, in some embodiments, A=S, O, or N. In someembodiments, D=H, —CH═CH—CH=E-, halides, cyano, sulfonates, alkyl chain,or trifluoromethyl. In some embodiments, E=H, —CH═CH—CH=D-, halides,cyano, sulfonates, alkyl chain, or trifluoromethyl.

In some embodiments, X¹ is a moiety conferring water solubility. In someembodiments, Y¹ is a tri-substituted aryl group. In some embodiments,the Y¹ (e.g., the tri-substituted aryl group) comprises a NHS-estermoiety (e.g., for protein linkage); an oligonucleotide; a peptide; afluorescence quencher; a pro-fluorophore; an alkyne (e.g., for clickchemistry); a triazene (e.g., from click reaction); the like, or acombination thereof. In some embodiments, Y¹ comprises an aldehyde; anamine (e.g., Fmoc protected), aminooxy, halogen (e.g., radio isotope);the like, or a combination thereof. In some embodiments, Z¹ is anoptionally substituted aryl. In some embodiments, Z¹ comprises aNHS-ester moiety; an oligonucleotide; a peptide; a fluorescencequencher; a pro-fluorophore; a biologically active acid labile compound;a prodrug comprising a phenolic functional group; releasable cargo; analkyne (e.g., for click chemistry); a triazene (e.g., from clickreaction); a polymerization residue (e.g., epoxide, polystyrene,alpha-beta-unsaturated ester acrylate, polyacrylamide, an amine, etc.),the like, or a combination thereof. In some embodiments, Z¹ comprises analdehyde; an amine (e.g., Fmoc protected), aminooxy, halogen (e.g.,radio isotope); the like, or a combination thereof.

In some embodiments, X¹ may comprise a functional group that conferswater solubility. In some embodiments, X¹ comprise a moiety of theformula —R¹-Q¹, wherein R¹ is C₁₋₆ alkylene, and Q¹ is sulfate,sulfonate, phosphate, a quaternary ammonium cation, or an alkyl, aryl orpropargylic containing moiety that can facilitate coupling to otherazides via [3+2] cycloaddition chemistry. In some embodiments, X¹ is amoiety of the formula —R¹-Q¹, wherein R¹ is an alkane, e.g., C₁₋₆alkylene. In some embodiments, Q¹ is sulfate (e.g., —(O)_(n)SO₃R^(a),where n is 0 or 1, and R^(a) is C₁₋₆ alkyl or typically H), phosphate(e.g., —(O)_(n)PO₃R^(a), where n is 0 or 1, and R^(a) is C₁₋₆ alkyl ortypically H), or a quaternary ammonium cation (e.g.,—[NR^(a)R^(b)R^(c)]⁺, where each of R^(a), R^(b), and R^(c) isindependently H or C₁₋₆ alkyl). As used herein, the term “alkyl” refersto a saturated linear monovalent hydrocarbon moiety of one to twelve,typically one to six, carbon atoms or a saturated branched monovalenthydrocarbon moiety of three to twelve, typically three to six, carbonatoms. Examples of alkyl groups include, but are not limited to, methyl,ethyl, n-propyl, 2-propyl, tert-butyl, pentyl, and the like. The term“alkylene” refers to a saturated linear divalent hydrocarbon moiety ofone to twelve, typically one to six, carbon atoms or a branchedsaturated divalent hydrocarbon moiety of three to twelve, typicallythree to six, carbon atoms. Examples of alkylene groups include, but arenot limited to, methylene, ethylene, propylene, butylene, pentylene, andthe like.

Triazabutadiene molecules of the present invention are readily solublein water. In some embodiments, the solubility of the triazabutadienemolecules in water is at least 23 g/L of water (50 mM). In someembodiments, the triazabutadiene molecules are stable in pH 7.4phosphate buffer. The phosphate buffer solutions are commerciallyavailable or can be prepared, for example, as described inhttp://cshprotocols.cshlp.org/content/2006/1/pdb.rec8247. In someinstances, the half-life of the triazabutadiene molecules of the presentinvention in pH 7.4 phosphate buffer solution is at least 24 hours.

Stability of the triazabutadiene molecule can be measured in variousways. In some embodiments, stability is measured by the half-life of themolecule (or the half-life of the molecule in a particular buffer at aparticular pH). In some embodiments, the molecule has a half-life of atleast 12 hours in a pH 7.4 buffer. In some embodiments, the molecule hashalf-life of at least 24 hours in a pH 7.4 buffer. In some embodiments,the molecule has half-life of at least 36 hours in a pH 7.4 buffer. Insome embodiments, the triazabutadiene molecule has a half-life of atleast 8 hours. In some embodiments, the triazabutadiene molecule has ahalf-life of at least 10 hours. In some embodiments, the triazabutadienemolecule has a half-life of at least 12 hours. In some embodiments, thetriazabutadiene molecule has a half-life of at least 20 hours. In someembodiments, the triazabutadiene molecule has a half-life of at least 24hours. In some embodiments, the triazabutadiene molecule has a half-lifeof at least 30 hours. In some embodiments, the triazabutadiene moleculehas a half-life of at least 36 hours. The present invention is notlimited to the aforementioned examples of stability measurements.

Without wishing to limit the present invention to any theory ormechanism, it is believed that the triazabutadiene molecules of thepresent invention are advantageous because the triazabutadiene moleculescan be easily modified (e.g., various different functional groups can beeasily used as X¹, Y¹, or Z¹ (see FIG. 1). And, the release of thediazonium species following triazabutadiene molecule breakdown (viacertain mechanisms, as described below) provides a functional group thatcan be taken advantage of in various applications. Also, it may beconsidered advantageous that the breakdown of the triazabutadienemolecule is irreversible.

II. Cleavage of Triazabutadiene Molecules

a. Water and/or Low pH

The present invention shows that triazabutadiene molecules may breakdown in the presence of water to generate reactive aryl diazoniumcompounds. For example, FIG. 2A shows that triazabutadiene molecules ofthe present invention can undergo decomposition to diazonium salts(reactive aryl diazonium compounds) and cyclic guanidine species. Aryldiazonium compounds can react with electron-rich aryl rings (e.g., arylspecies wherein the bond of interest is a nitrogen-carbon bond; indoles,anilines, phenol-containing compounds such as resorcinol or tyrosine,etc.) to form stable azobenzene linkages (e.g., an aryl azo dye, e.g.,Sudan Orange). (Note the present invention is not limited to theaforementioned phenol-containing species. In some embodiments, imidazolecompounds (e.g., purine bases like guanine) may be used in lieu of aphenol-containing compound.) The diazonium species may not necessarilyreact with an electron-rich aryl rings compound (e.g., phenol species),for example if a phenol species is not present. The diazonium speciesmay irreversibly extrude nitrogen gas to generate an aryl cation, whichwill rapidly be quenched by solvating water, thus synthesizing a newphenolic compound (e.g., HO-Ph, wherein Ph refers to the phenyl ring);thus, the diazonium portion of the triazabutadiene molecule may functionas a masked hydroxyl group.

In some embodiments, the triazabutadiene molecules are acid labile,e.g., unstable at particular pH levels (see FIG. 2B). For example,decreases in pH increase the rate at which the triazabutadiene moleculesbreak down (the half life of the molecule decreases). In someembodiments, the triazabutadiene molecules are unstable at low (lowered)pH levels (e.g., lowered pH as compared to a particular pH that themolecule may be stored at, e.g., a pH wherein the molecule has aparticular desired half life). Low pH levels, in some example, may be asub-physiological pH (7.4 or less). In some embodiments, thetriazabutadiene molecules are (more) unstable at pH 7.0 or less, pH 6.8or less, pH 6.5 or less, pH 6.2 or less, pH 6.0 or less, pH 5.8 or less,pH 5.6 or less, pH 5.5 or less, pH 5.2 or less, pH 5.0 or less, etc.

The term ‘low pH” may refer to several different pH levels. Since thefunctional groups attached to the molecule (e.g., see X¹, Y¹, Z¹ ofFormula I) affect the stability of the molecule (as well as watersolubility), the pH that is necessary to increase the rate of breakdownof the triazabutadiene molecule (e.g., the “lowered pH”) may bedifferent for different molecules. In some embodiments, the low pH is apH of 7.4 or less. In some embodiments, the low pH is a pH of 7.2 orless. In some embodiments, the low pH is a pH of 7.0 or less. In someembodiments, the low pH is a pH of 6.8 or less. In some embodiments, thelow pH is a pH of 6.6 or less. In some embodiments, the low pH is a pHof 6.6 or less. In some embodiments, the low pH is a pH of 6.6 or less.In some embodiments, the low pH is a pH of 6.5 or less. In someembodiments, the low pH is a pH of 6.4 or less. In some embodiments, thelow pH is a pH of 6.2 or less. In some embodiments, the low pH is a pHof 6.0 or less. In some embodiments, the low pH is a pH of 5.8 or less.In some embodiments, the low pH is a pH of 5.5 or less. In someembodiments, the low pH is a pH of 5.0 or less.

In some embodiments, the triazabutadiene molecules can break downwithout the presence of the low pH (the molecules have half lives);however, in some embodiments, a lowered pH enhances the reaction (e.g.,increases the rate of reaction). As such, a low pH may or may not beused with the molecules and/or methods of the present invention. In someembodiments, the triazabutadiene molecule has a half-life of no morethan 1 hour in a pH 7.4 aqueous solution. In some embodiments, thetriazabutadiene molecule has a half-life of no more than 30 minutes in apH 7.4 aqueous solution. In some embodiments, the triazabutadienemolecule has a half-life of no more than 15 minutes in a pH 7.4 aqueoussolution.

The present invention also features methods of breaking downtriazabutadiene molecules. In some embodiments, the method comprisessubjecting the molecule to water. In some embodiments, the methodcomprises subjecting the molecule to a low pH (e.g., a low pH that isappropriate for the molecule, e.g., a lowered pH that increases the rateat which the triazabutadiene molecule breaks down).

In some embodiments, the reaction of the triazabutadiene molecule to thediazonium species occurs in water within 10 seconds minutes. In someembodiments, the reaction of the triazabutadiene molecule to thediazonium species occurs in water within 30 seconds minutes. In someembodiments, the reaction of the triazabutadiene molecule to thediazonium species occurs in water within 1 minute. In some embodiments,the reaction of the triazabutadiene molecule to the diazonium speciesoccurs in water within 5 minutes. In some embodiments, the reaction ofthe triazabutadiene molecule to the diazonium species occurs in waterwithin 10 minutes. In some embodiments, the reaction of thetriazabutadiene molecule to the diazonium species occurs in water within15 minutes. In some embodiments, the reaction of the triazabutadienemolecule to the diazonium species occurs in water within 20 minutes. Insome embodiments, the reaction of the triazabutadiene molecule to thediazonium species occurs in water within 25 minutes. In someembodiments, the reaction of the triazabutadiene molecule to thediazonium species occurs in water within 30 minutes. In someembodiments, the reaction of the triazabutadiene molecule to thediazonium species occurs in water within 45 minutes. In someembodiments, the reaction of the triazabutadiene molecule to thediazonium species occurs in water within 60 minutes.

In some embodiments, the diazonium species may be visuallydifferentiated from the triazabutadiene species, e.g., the diazoniumspecies is visually distinct (e.g., a different color) from thetriazabutadiene molecule. If applicable, in some embodiments, the arylazo dye may be visually differentiated from the triazabutadiene speciesand the diazonium species, e.g., the aryl azo dye is visually distinct(e.g., a different color) from the triazabutadiene species and thediazonium species.

Given the possibility that the aryl azo dye is visually distinct fromthe triazabutadiene molecule (and/or the diazonium species), the presentinvention also features methods of producing a visually detectablemolecule. In some embodiments, the method comprises providing atriazabutadiene molecule according to the present invention andsubjecting the triazabutadiene molecule to water and/or a low pH (orlight as discussed below, or light and low pH, etc.). The low pH (orlight, or light and low pH, etc.) initiates (e.g., increases the rateof) the irreversible reaction to produce the diazonium species and thecyclic guanidine species. As previously discussed, the diazonium speciesmay be visually distinct from the triazabutadiene molecule; thereforethe reaction produces a visually detectable molecule.

b. Reductive Cleavage

Other mechanisms may be used to break down triazabutadiene molecules ofthe present invention. For example, in some embodiments, reducingconditions increase the rate at which the triazabutadiene moleculesbreak down. Thus, the present invention also features methods ofreductive cleavage of triazabutadiene molecules. For example,triazabutadiene molecules (e.g., triazabutadiene scaffolds) may bereadily cleaved using reducing agents such as but not limited to sodiumdithionite (sodium hydrosulfite) (Na₂S₂O₄) (see FIG. 3). In someembodiments, the reducing agent comprises lithium aluminum hydride,sodium borohydride, or the like.

In some embodiments, electrochemical reduction may be used in accordancewith the present invention. Reductive cleavage of the triazabutadienemolecules provides a urea functionality and a terminal aryl triazene(see FIG. 3). In some embodiments, the aryl triazene is further reducedin the presence of excess reducing agent (e.g., sodium dithionite). Insome embodiments, the reduction can be observed visually by the changein color of a solution. For example, there may be a subtle change ofyellows that results from a loss of a shoulder in UV/vis spectrum.

In some embodiments, the ratio of the concentration of thetriazabutadiene to the reducing agent is about 1:1. In some embodiments,the ratio of the concentration of the triazabutadiene to the reducingagent is about 1:2. The present invention is not limited to theaforementioned ratios. For example, in some embodiments, the ratio ofthe concentration of the triazabutadiene to the reducing agent is about2:3, 4:5, etc. The present invention is not limited to theaforementioned ratio of concentrations.

In some embodiments, the reduction can occur within about 10 minutes,within about 15 minutes, within about 20 minutes, within about 25 min,within about 30 min, etc., at room temperature. Without wishing to limitthe present invention to any theory or mechanism, it is believed thatreductive cleavage of the triazabutadiene molecules is advantageousbecause it can occur rapidly (e.g., within 10 minutes, within 15minutes). Also, the triazabutadiene molecules that are highly stable inacid (e.g., a p-CN derived triazabutadiene) may still be susceptible toreducing conditions.

In some embodiments, reductive cleavage of triazabutadiene molecules mayalso be used to cleave unreacted triazabutadienes that did not undergodiazonium formation/reaction chemistry that is associated with a drop inpH (or other mechanism) as described above (a sort of quench for the pHchemistry).

c. Light-Initiated Cleavage

In some embodiments, light increases the rate at which thetriazabutadiene molecule breaks down (into the cyclic guanidine speciesand the diazonium species) (see FIG. 4A). The present invention featurestriazabutadienes that, upon photo-irradiation, may be rendered morebasic in a reversible fashion. Referring to FIG. 4B, for reference, aprotecting group of a masked base may decompose to reveal a basicnitrogen atom upon exposure to light. Or, a basic nitrogen atom of amolecule obscured by a steric wall may be reversibly swung away in aphotochemically-triggered manner. The present invention shows theintrinsic basicity of a nitrogen-containing functional group may bealtered by a photochemical event.

Referring to FIG. 5A, in some embodiments, triazabutadiene molecules ofthe present invention may readily photoisomerize to a more reactiveZ-form. As an example, an aqueous solution of Compound A was irradiatedwith a simple hand-held UV lamp (“365 nm,” measured at 350 nm).Consumption of Compound A was observed after only a few hours. Thenon-irradiated reaction under similar conditions was stable for days aspartial degradation rapidly renders the solution mildly basic. Withoutwishing to limit the present invention to any theory or mechanism, itwas hypothesized that if a two-electron process were happening, thenCompound A-Z would be more basic than Compound A-E. A 1.0 N NaOHsolution of Compound A was treated with light. At pH 14, Compound A wasstable for weeks in the dark; it was surprisingly discovered that nearcomplete consumption of starting material after 20 hours of constantirradiation occurred. NMR analysis of samples post-irradiation showedcyclic guanidine Compound B. Evidence of a benzene diazonium species orphenol/azobenzene products derived therefrom was not observed. Benzenediazonium ions also absorb UV light to expel nitrogen and generate abenzene radical. In order to resolve if the initial cleavage undergoes aradical homolytic mechanism versus a two-electron heterolytic mechanism,a trapping experiment using resorcinol was conducted. (Resorcinol waschosen because it can serve a dual role as a radical scavenger and atrap benzene diazonium species that could be formed.) An excess ofresorcinol was added to a pH 9 borate-buffered solution of Compound Aand the mixture was irradiated with light. The known azobenzene, SudanOrange G, was formed in a 65% yield (versus 4% for the non-irradiatedreaction). Derivatives of Compound A were made to examine the effects ofelectronic perturbations on the light-induced degradation. Electrondeficient aryl rings are more stable at lower pH, and this trendgenerally holds true for the photochemical reactions as well. A bufferedborate solution was chosen due to its alkaline nature and lack ofcomplicating signals in the NMR experiment. Compounds C-E all haveabsorption spectra that are well within the range of the UV lamp (seeFIG. 4C(ii)). Both m-NO₂ (Compound C) and p-CN (Compound D) had similarrates of reaction, both slower than Compound A. To rule out othereffects associated with possible heating or interactions of the buffer,p-NO₂ derivative Compound E was irradiated because of its significantlyred-shifted spectrum. Compound E absorbed in a range that was notirradiated with the UV lamp and as such was recalcitrant to degradation(see FIG. 5A(iii)).

As previously discussed, poorly (or non-) buffered aqueous solutionscould become more basic as a function of time due to the degradation toCompound B and the aryl diazonium species. Without wishing to limit thepresent invention to any theory or mechanism, it is believed that thecause of the increase in pH is Compound B, which acts as a base. It wasfound that reactions slowed and eventually stopped once the pH had risento around 9. Without wishing to limit the present invention to anytheory or mechanism, it was hypothesized that by driving the reaction tocompletion with light, it would be possible to increase the pH beyondthis dark-reaction imposed wall (analogous FIG. 4B(ii)). Using NMR and apH meter, it was observed that the pH of a solution of Compound Airradiated with UV light rose in a time-dependent manner.

In an effort to examine the rate order for the pH-increasing reactionmore carefully, in situ, real-time pH measurements were acquired.Compound A was dissolved in water and the pH of the solution wasadjusted to 9 such that it would not form Compound B in the absence oflight. Upon exposing the solution to 350 nm light, it was surprisinglydiscovered that the solution rapidly spiked up to a pH of ˜10 over thecourse of several minutes, and only upon much longer exposure slowlybecame more basic. This spike was not at all consistent with the modelof the pH increase being solely linked to the concentration of CompoundB being generated. Moreover, previous NMR studies showed that much moretime was required to afford a pH change commensurate with this apparentlevel of degradation.

Without wishing to limit the present invention to any theory ormechanism, it was hypothesized that the rapid pH increase that wasobserved was not attributed to Compound B, but instead a result of the Zisomer being significantly more basic than the E isomer (see FIG. 5B(i).A sample was irradiated and then the light was turned off once the pH ofthe solution started to increase noticeably. As the sample thermallyreverted to the more stable E form, the pH of the solution dropped aswell (see FIG. 5B(ii)). The experiment was repeated with increasingtimes of irradiation, and a saw-tooth pattern was obtained. The processwas not completely reversible due to some degradation to Compound B.Indeed, triazabutadiene Compound A can serve a dual role of being aphoto-masked base (see FIG. 4B(i)), and a base whose intrinsicfunctional group properties are altered photochemically (FIG. 4B(iii)).

This phenomenon via an isomerization-induced pK_(b) change wassurprisingly discovered by the inventor. Without wishing to limit thepresent invention to any theory or mechanism, unlike the case whereHecht's compound is rendered basic upon irradiation by way of moving ofa steric wall (see FIG. 4B(ii), it is unlikely that steric factors playa significant role in this chemistry, especially in water. It ispossible that the E-isomer has alternating “non-π involved” lone pairsof electrons, whereas the Z-isomer has two adjacent “non-π involved”lone pairs of electrons (see FIG. 5C). The electronic repulsion fromthese renders N1 much more electron rich in Z-isomer and thus a strongerLewis base.

Referring to FIG. 5A(iii), Compound C and Compound D were examined in aneffort to find a base that was reversibly basic but also more resistantto degradation. In both cases, a slow subtle change to the pH wasobserved, but none as dramatic and rapid as Compound A. Without wishingto limit the present invention to any theory or mechanism, it isbelieved that this may be due to factors such as (a) faster thermalisomerization to the E isomer such that a build up of the Z isomer isnot possible; (b) the electron-deficient triazabutadienes are less basicto begin with, so a transition is not observable in the operating pHrange.

It is possible that Compound A may be useful as a photo-catalytic basein the context of organic reactions. With limited solubility in all butDMSO, the stability of Compound A was tested. As noted previously,Compound A is quite stable to an excess of acetic acid in DMSO, showingonly 12% degradation over 14 hours at room temperature. Upon irradiationwith light, Compound A in presence of acetic acid completely fell apartover the same time frame. To confirm that this was due to the acid, asolution of Compound A (in pure DMSO) was irradiated. After four hoursof constant irradiation in acid-free DMSO, an E:Z ratio of nearly 50:50was observed. Moreover, unlike in water, the thermal reversion from Z toE is slow in pure DMSO with a half-life on the order of days.Attributing this to lack of protonation, a control in MeOD was run, anda first-order thermal isomerization was observed with a rate of 3×10⁻⁵s⁻¹ (t_(1/2)˜6.4 hours), in addition to some degradation to Compound B.

Referring to FIG. 5D, due to the limited organic solubility of CompoundA, Compound F (FIG. 5D(i)) was synthesized. With Compound F, a similarlight-induced acid sensitivity was observed in DMSO (and slow thermalisomerization). Based on the apparent pK_(b) of Compound F, pK_(a) werematched to condensation substrates. A Henry reaction between nitroethane(Compound G) and p-nitrobenzaldehyde (H) was chosen to demonstrate thevirtues of Compound F (FIG. 5D(ii)). The reaction between Compound G andCompound H occurred rapidly at room temperature in a light and catalystdependent manner (FIG. 5D(iii)). The reaction with 25 mole % Compound Fin the absence of light was exceedingly slow. Likewise, the reactionwith light but no catalyst also failed to proceed. The cyclic guanidinewas not observed during a post-reaction analysis of the components froma 25 mole % Compound F run, indicating that the Z-isomer of Compound Fis likely to be the catalytically active species in solution. Slowthermal isomerization back to the E-isomer in aprotic organic solventstogether with a fast overall reaction attempts to adjust the reactionrate prior to consumption of Compound H. Interestingly, the reactioncatalyzed with Compound F was significantly faster than the samereaction reported by Hecht. This may provide evidence that Compound F-Zis more basic than Hecht's blocked trialkylamine.

As previously discussed, the present invention features methods ofbreaking down triazabutadiene molecules by subjecting the molecule tolight. The light may, for example, include wavelengths of about 400 nm.The present invention is not limited to wavelengths of 400 nm or about400 nm. For example, in some embodiments, the wavelength is from 350 nmto 400 nm (e.g., 370 nm). In some embodiments, the wavelength is from360 nm to 410 nm. In some embodiments, the wavelength is from 330 nm to420 nm. In some embodiments, the wavelength is from 340 nm to 430 nm. Insome embodiments, the method comprises subjecting the molecule to a lowpH and to light.

As previously discussed, light-promoted reactivity andlight-facilitating E/Z isomerization has been observed. In someembodiments, a system such as a UV-LED pen may be used for thesereactions, however the present invention is not limited to a UV-LED penand may utilize any appropriate system. The UV-LED pens may allow forrelatively narrow bandwidth irradiation of these compounds (but are notlimited to these bandwidths). The color of the bulk material shifts as aresult of electronic perturbations to the aryl azide starting material.For example, nitro derivative Compound 6e of FIG. 6G is rust-red, versusan orange phenyl Compound 6c of FIG. 6F) and yellow-orange methoxyCompound 6d of FIG. 6G. It may be possible for selective irradiation ofa complex mixture in an orthogonal sense. These experiments may beperformed in basic aqueous solutions to maintain the solvationproperties of water while also preventing the degradation pathwaystemming from protonation. These experiments are not limited to basicaqueous solutions.

Without wishing to limit the present invention to any theory ormechanism, it may be considered advantageous that the breakdown of thetriazabutadiene molecule is irreversible.

III. Synthesis of Water-Soluble Triazabutadiene Molecules andExperimental Examples

Synthesis of 1-mesityl-1-H-imidazole: To a solution of2,4,6-trimethylaniline (1.35 g, 10.0 mmol) in methanol (15 mL)

added a solution of glyoxal (40%) (1.14 mL, 40% in water, 10. mmol). Themixture was stirred at room temperature until a solid formed.Thereafter, solid ammonium chloride (1.07 g, 20 mmol), formaldehyde(37%) (1.6 mL 37% in water, 60. mmol) and methanol (40 mL) were added,and the mixture was heated to reflux for one hour. After the hour,phosphoric acid (1.4 ml of an 85% solution) was added drop wise and themixture was refluxed for an additional eight hours. Upon cooling to roomtemperature ice (30 g) was added and the solution was brought to a pH of9 with potassium hydroxide (40% in water). The following mixture wasextracted repeatedly with diethyl ether. The ether phase was dried overmagnesium sulfate and solvent removed in vacuo to form a brown solidwhich was filtered and washed with hexanes to give the product (0.785 g;42%). 1H NMR (500 MHz, CDCl3): δ 7.45 (t, J=1.1 Hz, 1H), 7.25 (t, J=1.1Hz, 1H), 6.99 (dp, J=1.3, 0.7 Hz, 2H), 6.91 (t, J=1.3 Hz, 1H), 2.36 (t,J=0.7 Hz, 3H), 2.01 (t, J=0.6 Hz, 6H). 13C NMR (126 MHz, CDCl3) δ138.80, 137.47, 135.42, 133.40, 129.55, 128.96, 120.02, 21.03, 17.33.(see Liu, J. et al. Synthesis 2003, 17, 2661-2666).

Synthesis of 3-(1-mesityl-1H-imidazol-3-ium-3-yl) propane-1-sulfonate(see FIG. 6F): To a solution of 1-mesityl-1-H-imidazole (1.00 g, 5.36mmol) in toluene (30 mL) was added 1,3-propanesultone (1.00 g, 8.18mmol) and the mixture was heated to reflux overnight. The mixture wasallowed to cool to room temperature and the off-white precipitatecollected by filtration. The precipitate was further washed with diethylether and dried using a vacuum oven to yield a solid (1.40 g; 84%). 1HNMR (500 MHz, D2O): δ 8.92 (t, J=1.6 Hz, 1H), 7.75 (t, J=1.8 Hz, 1H),7.49 (t, J=1.8 Hz, 1H), 7.06 (q, J=0.8 Hz, 2H), 4.44 (t, J=7.1 Hz, 2H),2.39-2.31 (m, 2H), 2.25 (s, 3H), 1.96 (s, 6H). 13C NMR (126 MHz, D2O) δ141.42, 136.54, 134.64, 130.74, 124.34, 123.00, 48.18, 47.17, 25.03,20.17, 16.29.

Synthesis of Potassium3-(3-mesityl-2-(phenyltriaz-2-en-1-ylidene)-2,3-dihydro-1H-imidazol-1-yl)propane-1-sulfonate (see FIG. 6G): To a slurry of3-(1-mesityl-1H-imidazol-3-ium-3-yl)propane-1-sulfonate (50 mg, 0.16mmol) in dry THF (6 mL), was added a solution of phenyl azide in THF(0.16 mL, 1 M, 0.16 mmol). To the solution was added KO-t-Bu (24 mg,0.21 mmol) in one portion and the resulting mixture was stirred underargon for 4 hours. Hexanes (1 mL) was then added and the reactionmixture was filtered. The solvent was removed and the residue taken upin a minimal amount of DCM and on trituration with hexanes, pure productwas obtained by filtration as a yellow powder (61 mg, 81%). 1H NMR (500MHz, DMSO-d6) δ 7.32 (d, J=2.4 Hz, 1H), 7.07-7.02 (m, 4H), 6.99-6.94 (m,1H), 6.84 (d, J=2.4 Hz, 1H), 6.51-6.47 (m, 2H), 4.09 (t, J=7.1 Hz, 2H),2.34 (s, 3H), 2.12-2.04 (m, 2H), 1.95 (s, 6H). 13C NMR (126 MHz,DMSO-d6) δ 152.19, 151.13, 137.94, 136.15, 134.31, 129.31, 128.60,125.26, 120.90, 117.61, 117.24, 48.52, 45.05, 25.80, 21.06, 17.95.

Using the procedures described herein, the p-methoxy and p-nitro analogs(from the p-MeO aryl azide and p-NO2 aryl azide) were also prepared.

For decomposition experiments, buffers were made to the appropriate pHin a 9:1 mix of H2O:D2O. These solutions were added to the compoundbeing assayed such that the buffer capacity was at least 10 fold theconcentration of the compound. Some experiments used 5 mg compound in0.5 mL of buffer. These were immediately inserted into an NMR instrumentand scans were taken at even time intervals to calculate the half-lifeof the compound based on integration.

As another non-limiting example, an azide (e.g., NHS-azide) toN-heterocyclic carbene (NHC) route may be used to synthesizetriazabutadiene molecules (e.g., see FIG. 6C). For example, as shown inCompound 4 of FIG. 6D, a triazabutadiene molecule was synthesized fromdimethyl imidazole derived NHC and phenyl azide. When thetriazabutadiene molecule (Compound 4) was treated with methanolic HCL, arapid color change occurred. This change was confirmed to coincide withdiazonium formation by trapping the reactive species with resorcinol toprovide known diazo dye Sudan Orange G. When the triazabutadienemolecule (Compound 4) was treated with the much less acidic acetic acid,the same product was obtained. Compound 4 was not water-soluble.

To render the triazabutadiene water-soluble, methyl imidazole wasalkylated with propane sultone to provide the Zwitterionic NHC precursorCompound 5a (see FIG. 6E). Formation of the NHC under basic conditionsin the presence of phenyl azide yielded the highly water solubleCompound 6a (see FIG. 6E). Compound 6a was highly colored, so its pHdependence was studied using UV/Vis. The reactions were not only pH-,but also scan-frequency dependent. Upon finding this, the stability ofCompound 6a was studied in D2O in the dark using NMR. Even in the darkit was unstable, but not in the diazonium-forming way. Both Compound 6band a more hindered mesityl (Mes) substituted Compound 6c (see FIG. 6E)were synthesized to stabilize what was initially considered to be arearrangement pathway that could be blocked by steric repulsion.Compound 6c was the most stable of the three (less than 10% consumedafter 24 hours versus 50% for Compound 6a and Compound 6b). It is notyet clear that the hypothesis of a simple rearrangement was correct.Dissolution in 0.1 N NaOH rendered all compounds stable (no detectabledegradation after 24 hours in the dark).

As mentioned above, Compound 6c was reasonably stable in pure D20. Uponadjusting the pH to 5 with HCl, a rapid initial consumption of Compound6c to Compound 7 (see FIG. 6E) and a benzenediazonium salt was noted.After this initial burst of reactivity, a slowing and apparent arrestingof the reaction was noted. At this pH the hydronium was the limitingreagent. All future reactions were run in buffers with a buffer capacitysufficient to maintain a large excess of hydronium ions. The experimentswere performed in 90:10 H2O:D2O buffered solutions to minimizeconsiderations of pH vs. pD. The decomposition to diazonium salts andCompound 7 was measured as a function of pH in phosphate/citrate buffersfrom pH 4-7 and in a phosphate buffer from pH 6-8. All runs providedlinear correlations of concentration and time, indicating a pseudo-zeroorder reaction (first order with respect to hydronium ion with a largeexcess of hydronium ions). While the peaks for Compound 7 remainedconstant, the peaks associated with Compound 6c drifted downfield as thereaction progressed. This drifting was highly reproducible acrosssamples and buffers, but the underlying cause is not understood at thistime. A sigmoidal correlation between rate and buffer pH centered at pH6 was obtained. When resorcinol was not added to consume the diazoniumspecies, 4-phenylazophenol (Compound 8) was observed (see FIG. 6F).Compound 8 came from the decomposition of one diazonium ion to phenolfollowed by reaction with a second diazonium ion. The instability ofCompound 6c in a pH 7 phosphate buffer was surprising given thestability in D2O. Compound 6c was tested in a non-buffered 90:10 H2O:D2Osolution and observed only >7% after 6 hours.

To further examine the reactivity of this class of compounds, variantsCompound 6d and Compound 6e were synthesized (see FIG. 6G). It washypothesized that the p-methoxy and p-nitro analogs (Compound 6d andCompound 6e, respectively) would display different reactivity profiles.It was observed that in pure D20, 26% of Compound 6d was consumed after24 hours in the dark at room temperature as compared with Compound 6e,which was stable to within the detection limit of NMR. Preliminary datashows that Compound 6d undergoes decomposition to the diazonium speciesmore rapidly than Compound 6c in pH 5, 6, and 7 phosphate/citrate buffer(rates of 2.0×10-5, 1.0×10-5, and 0.53×10-5 M/s, respectively). Uponattempting the same study with Compound 6e it rapidly precipitated outof solution across the same pH range. After collecting the precipitateand dissolving it in deuterated methanol, no change was observed from asample of Compound 6e that had never been exposed to a bufferedsolution. Treatment of this methanolic Compound 6e with HCl led to animmediate color change and diazonium formation was confirmed by trappingwith resorcinol. It is possible that: 1) that the sodium salt ofCompound 6e is much less soluble than the potassium salt; or 2) withdifferent solvating ions present the sulfonate interacts with theelectron-poor N2 nitrogen atom of the triazabutadiene to breakconjugation and form an insoluble complex (this is backed by areversible color change of the starting rust-red solid, to the lightyellow precipitate). Note that the p-nitrobenzenediazonium salts arereported to have the best labeling efficiency of tyrosine residues onproteins.

The influence of solvated ions on reactivity was studied. In water, or aheavy water/water mixture, a near-zero rate of diazonium salt formationwas observed, yet in solutions buffered to pH 7 and even pH 7.4 anincrease in the reaction rate was observed. To assess the role of theanionic component, the reaction in the presence of a range of bufferswhile holding the pH constant will be observed. Buffers include but arenot limited to those expected to have the most diverse properties, e.g.,MES, a Zwitterionic morpholino sulfonic acid, and imidazolium chloride,the conjugate acid of a mild base, can both buffer a solution at pH 6.5,but ionic species in solution would be dramatically different. Themetals in solution could well be acting as Lewis acids to activate ourmolecule. A range of metal halide salts dissolved in pure water atvarying concentrations will be screened.

Note that all of the compounds in the 6 series (FIG. 6E, FIG. 6F, andFIG. 6G) have a built-in sulfonate to confer solubility. It is possiblethat this functional group could be serving an important role byeffecting the localization of metals, directing them to interact withthe nitrogen atoms of the triazabutadiene and thus alter the reactivityof the compound. This may be happening with Compound 6e to such anextreme that the compound is no longer soluble. This concept of adirected metal binding on triazabutadienes was observed, albeit in anorganic environment. Referring to FIG. 6H, to study the role of the sidechain, the imidazole core will be alkylated (see Compound 9) with eitherbutane sultone to provide imidazolium (Compound 10) and triazabutadiene(Compound 11), or a dialkyl aziridinium salt to provide the analogousCompound 12 and Compound 13 which invert the expected charge on theside-chain. The extra methylene in Compound 11 as compared with Compound6 may alter the way that the side-chain bites back on thetriazabutadiene. The tertiary amine will be protonated at physiologicalpH and as serve to invert the charge of the side arm. Without wishing tolimit the present invention to any theory or mechanism, a potentialbonus of Compound 13 is that the basic nitrogen may help localize thiscompound in the most acidic subcellular compartments much likeLysoTracker™ dyes.

Regarding the role of mesityl group in reactivity, it is possible that afunction of the mesityl in triazabutadiene reactivity is to provide asteric wall to prevent side reactions. The NMR of Compound 6c (see FIG.6F) shows a tale of two hydrogen atoms on the imidazole ring. Withoutwishing to limit the present invention to any theory or mechanism, it isbelieved that because the ortho methyl groups prevent coplanar arylrings, the mesityl group is unlikely to sit in conjugation with theimidazole, but the highly differentiated chemical environments might beexplained by: 1) the mesityl π-system deshielding the adjacent hydrogenatom, and 2) the aryl ring having an inductive effect. Changing thep-methyl of the mesityl to electron donating and withdrawing groups mayallow the adjustment of the electronic parameters without disrupting thesteric bulk.

Referring to FIG. 7A, in some embodiments, synthesis may be performedwith known p-azido dimethyl aniline (Compound 14) because it may lead toa wide range of substituted compounds. From imidazole (Compound 15) onecan alkylate with 1,3-propanesultone to provide NHC precursor Compound16, or prior to that one can treat with an NHC to access the wealth ofdiazonium chemistry to provide Compound 17 in all of its forms.Solvolysis in water or alcoholic solvent may provide a phenol or arylether, and copper mediated Sandmeyer-type chemistry may afford cyano,nitro or halogenated aryl species. From imidazolium Compound 16Staudinger chemistry followed by aniline alkylation may provide Compound18, or traceless Staudinger-Bertozzi ligation may yield Compound 19.These substrates cover a range of Hammett values while also providing anadditional site of attachment to proteins, fluorophores, surfaces, etc.

Referring to FIG. 7B, regarding the role of intramolecular hydrogen bondacceptors/donors in reactivity, it may be possible to synthesize aseries of triazabutadienes with hydrogen bond donors that possess arange of pKa values (Compounds 20-22). In addition to H-bond donors, itmay be possible to synthesize a series of internal bases (Compounds23-25). It may be possible that bases (e.g., dimethyl amine) positionednear the N1 nitrogen will favor protonation at N3 and thus make thetriazabutadiene less stable to acidic media. These compounds are allsynthetic targets given a strategy of coupling with aryl azides. Thedelicate triazabutadiene functional group is installed last under mildconditions. In addition to compounds that are designed toactivate/deactivate the N1 nitrogen, it may be possible to synthesize aseries of compounds where the N3 nitrogen in most likely to be affected(Compounds 26-28). An NHC with a hydrogen bond donor on a short arm wasmade. As in FIG. 7C, the synthesis of Compounds 26-28 from knownCompound 29 may start with either alkylation to a compound like Compound31 or reduction and protection to compound 30 followed by alkylation toCompound 32. If the mesityl is absolutely essential for a desiredreactivity profile, a H-bond donor/acceptor may be inserted on a methylgroup in the ortho position of the mesityl ring.

Referring to FIG. 7D, regarding intramolecular trapping of diazoniumspecies, it may be possible to synthesize triazabutadienes with adjacentfunctional groups that will rapidly consume the diazonium species. Forexample, Compound 33 contains an aryl ring, positioned ortho to themasked diazonium. The synthesis may start from a diazo transfer reactionto convert aniline Compound 34 to an aryl azide. Coupling with Compound5c (FIG. 6E) may complete the synthesis. It is possible that followingdiazonium unmasking an aromatic substitution reaction will occur toprovide benzocinnoline Compound 35. Because this reaction isintramolecular one might be able to use a non-activated ring, renderingthe ring electron rich. The methyl ether may serve as a site ofattachment to chemical cargos. A second type of intramolecular diazoniumtrap that could be employed is a beta keto ester that is also ortho tothe diazonium produced. Beta keto esters are known to react withdiazonium species through enol form, and can generate oxo-cinnolines,which are biologically active cores.

IV. Applications and Methods of Use of Triazabutadienes

The triazabutadiene molecules of the present invention may be utilizedfor a variety of purposes. For example, in some embodiments, thetriazabutadiene molecules of the present invention are utilized for acleavable linkage (e.g., chemoselectively-cleavable linkage) for use inbiological/complex settings where rapid, clean cleavage is of interest.In some embodiments, the triazabutadiene molecules are used for systemsincluding but not limited to drug delivery systems, protein-proteininteraction systems, pH environment detection systems, etc. Applicationsof these triazabutadienes may fall under one (or more) categories ofreactivity.

a. Diazonium Coupling Applications and Triazabutadiene Probes

Regarding diazonium coupling, the triazabutadiene molecules may be usedfor applications involving pH-dependent protein coupling. Generalexamples involve methods for detecting protein-protein proximity orprotein-protein interactions (in a sample). In some embodiments, themethod comprises providing a first protein, wherein the first protein isconjugated with a triazabutadiene molecule according to the presentinvention. The first protein may be introduced to a sample. In someembodiments, the triazabutadiene molecule encounters a low pH in thesample; in some embodiments, acid is added to the sample to lower the pHappropriately. As previously discussed, in the low pH environment, thetriazabutadiene molecule undergoes the irreversible reaction yieldingthe diazonium species and the cyclic guanidine species. As previouslydiscussed, the diazonium species is adapted to react with a phenolgroup; thus if there is a nearby protein with a tyrosine residue, thediazonium species may react with it yielding an azobenzene product(often colored, for example the dye, Sudan Orange G is an azobenzenecontaining dye) that is visually distinct from the triazabutadienemolecule and the diazonium species. As such, detection of the azo dyemay be indicative of proximity or interaction of the first protein andthe second protein. Thus, in some embodiments, the method comprisesadding a second protein to the sample, wherein a tyrosine of the secondprotein may react with the diazonium species. In some embodiments, thesecond protein is already in the sample. In some embodiments, a tyrosineor phenol species conjugated to the second protein. In some embodiments,the method comprises introducing to the sample a first antibody specificfor a first protein, wherein the first antibody is conjugated with atriazabutadiene molecule according to the present invention. In someembodiments, the method comprises introducing to the sample a secondantibody specific for a second protein. In some embodiments, the secondantibody comprises a tyrosine. In some embodiments, the second antibodyis conjugated with a phenol species. In some embodiments, the methodcomprises introducing an acid to the sample to appropriately lower thepH of the sample. As previously discussed, in the low pH environment,the triazabutadiene molecule undergoes the irreversible reactionyielding the diazonium species and the cyclic guanidine species. Aspreviously discussed, the diazonium species is adapted to react with aphenol group; thus if the phenol species is nearby, the diazoniumspecies may react with it yielding an azo dye that is visually distinctfrom the triazabutadiene molecule and the diazonium species. As such,detection of the azo dye may be indicative of proximity or interactionof the first protein and the second protein.

As a more specific example, the acid-labile reactivity oftriazabutadienes may be used to assist in work deducing interactionpartners between a virus and endosomally localized host proteins. Uponendosomal acidification a viral-bound diazonium species may be unmaskedand this may go on to react with Tyr-containing proteins that areassociating with the virus. It is possible that this system could beused to detect or trap an interaction that is relevant at a key point ofviral entry, e.g., the fusion of membranes. Herein are non-limitingexamples of synthesis of compounds that may be used in such systems,e.g., for modifying the viral surface. Lysine-reactive probes may beused to modify the surface of proteins. Referring to FIG. 8, bysynthesizing triazabutadiene Compound 36 bearing an N-hydroxysuccinimide(NHS) ester it may be possible to couple the compound to one of manyreactive Lys on the surface of the protein. As previously discussed, atriazabutadiene molecule may be attached to a viral protein (e.g., apurified viral protein). Then, a system such as a cell line (e.g.,mosquito cell line, human cell line, or even mosquitoes themselves) maybe infected with the viral protein. The infected system can be treatedappropriately. The azo dye (e.g., Sudan Orange) may “label” any proteinsthat interact with or are nearby the viral protein (in the low pHenvironment). The present invention is not limited to this example.Example 1 below describes the use of triazabutadienes in characterizingviral-host interactions.

Lys-NHS conjugation chemistry may work well on the basic side ofneutral, which may be beneficial for pH sensitive probes. Referring toFIG. 8 and FIG. 9, Compound 36 may be made in a straightforward fashionfrom NHC precursor Compound 5c (see FIG. 8) and an aryl azide. It ispossible that the steric congestion about the NHC may favor theunencumbered azide over the potentially reactive NHS ester. If the NHSester presents a problem during the synthesis it is possible to go intothe reaction with a carboxylate instead and follow that by a couplingwith N-hydroxysuccinimide. If electronically coupling the NHS ester tothe aryl system is detrimental to reactivity it is possible to considerinserting an alkyl or, if needed for additional solubility, polyethyleneglycol (PEG) linker. As an example, referring to FIG. 9, a monoclonalantibody (e.g., mouse anti-biotin) may be modified with Compound 36.Once the surface is decorated with triazabutadienes, the extent oflabeling may be quantified by coupling to resorcinol (or otherappropriate alternative) in a low pH solution and the extent ofmodification may be analyzed by mass spectrometry. This may show thenumber of reactive triazabutadienes. Following this analysis, afluorescent goat anti-mouse secondary antibody may be added, and then agel-shift assay may be used to show that the two antibodies arecovalently linked in a pH dependent manner.

As previously discussed, the present invention features triazabutadienesthat function as cross-linkers, e.g., cleavable cross-linkers. In someembodiments, the triazabutadiene cross-linkers allow for linkingcomponents via click chemistry, e.g., via copper-catalyzed azide-alkynecycloadditions. For example, if a clickable handle (e.g., a terminalalkyne handle) is disposed on the triazabutadiene, it can be used toundergo 1,3-dipolar cycloaddition with an azide handle on a differentcomponent (e.g., to yield a 1,4-disubstituted triazole) (see FIG. 10,which shows the click chemistry linking of an azide handle and an alkynehandle forming the 1,4-disubstituted triazole).

The use of triazabutadienes and click chemistry allows for the linkingof a wide range of compounds for either chemical or biologicalapplications. Note that in general, in order for the azide-alkynecycloaddition to occur, it must be activated with a Cu(I) source. Insome embodiments, the Cu(I) initiator can come from copper-halidereagents or Cu(II) sources that are reduced in situ. Cu(II) salts suchas CuSO₄ allow click chemistry to proceed in aqueous conditions withmild reducing agents such as sodium ascorbate (see FIG. 10). Cu(I)halide salts generally require a base/ligand to coordinate the metalinsertion and prevent oxidation. Without wishing to limit the presentinvention to any theory or mechanism, it is believed that copper clickchemistry is versatile as it can be performed in a wide range ofconditions. This may allow for tunability when it comes to finding theappropriate conditions for triazabutadiene functionalization.

Note that in some embodiments, the alkyne handle is disposed on thetriazabutadiene and said alkyne handle can react with an azide handle ona different component. The present invention is not limited to thealkyne handle being deposed on the triazabutadiene. In some embodiments,the azide handle is disposed on the triazabutadiene and said azidehandle can react with an alkyne handle on a different component. In someembodiments, both an alkyne handle and an azide handle is linked to thetriazabutadiene.

FIG. 11A shows non-limiting examples of structures of triazabutadiesadapted for click chemistry, e.g., Formula B and Formula C. In someembodiments, X¹ comprises an alkyne handle. In some embodiments, X²comprises an alkyne handle. In some embodiments, X¹ comprises an azidehandle. In some embodiments, X² comprises an azide handle. In someembodiments, the clickable triazabutadiene is according to Formula B,wherein X¹ comprises a terminal alkyne handle. In some embodiments, thetriazabutadiene is according to Formula C wherein X¹ comprises aterminal alkyne. In some embodiments, the triazabutadiene is accordingto Formula C wherein X² comprises a terminal alkyne handle. In someembodiments, the triazabutadiene is according to Formula C wherein bothX¹ and X² comprise a terminal alkyne handle. In some embodiments, A=S,O, or N; D=H, —CH═CH—CH=E-, halides, cyano, sulfonates, alkyl chain, ortrifluoromethyl. In some embodiments, E=H, —CH═CH—CH=D-, halides, cyano,sulfonates, alkyl chain, or trifluoromethyl. Note in some embodiments,the X¹ and Y¹ may be switched. For example, in some embodiments, A issulfur, and the alkyne is branched off of the other nitrogen.

As previously discussed, in some embodiments, the triazabutadienecomprises an alkyne handle. FIG. 11B shows TBD-6 and TBD-7, twonon-limiting examples of triazabutadienes with alkyne handles. In someembodiments, the alkyne handle is linked to the imidazole portion of thetriazabutadiene (TBD-6). In some embodiments the, the alkyne handle islinked to the aryl portion of the triazabutadiene (TBD-7). Note in someembodiments, the alkyne handle comprises a protection group (TBD-6comprises a protection group). In some embodiments, the protection groupcomprises chlorotripropylsilane (TPS); however, the protection group isnot limited to TPS. For example, in some embodiments, the protectiongroup comprises chlorotrimethylsilane (TMS-Cl), chlorotriethylsilane(TES-Cl), etc.

The synthesis of TBD-6 (from FIG. 11B) was performed as shown in FIG.12A beginning with silylation of propargyl bromide (Compound 13).(Protection of the alkyne was necessary due to the similar pK_(a) valuesof an alkyne (pK_(a)=25) and the proton on a NHC-salt (pK_(a)=21-24)).Propargyl bromide (Compound 13) was treated with n-butyllithium (n-BuLi)to deprotonate the terminal alkyne to render it nucleophilic so it wouldreact with Chlorotripropylsilane (TPS-Cl) yielding the silyl-protectedalkyne (Compound 14). The crude of Compound 14 was only partiallypurified by flash column chromatography, and was taken to reflux withN-mesitylimidazole in acetonitrile for 2 days to produce the imidazoliumsalt Compound 15. The coupling of Compound 15 and phenyl azide Compound8 took place in dry THF. KOtBu was added to generate the NHC to reactwith the azide and form Compound 6 in moderate yield. This reaction wasvery moisture sensitive and acquiring best yield in dry conditions. Thepresent invention is not limited to phenyl azide (Compound 8 of FIG. 12Aand FIG. 12B). In some embodiments, synthesis of clickabletriazabutadienes features alternative azide compounds such as Compound8′ (e.g., Compound) (see FIG. 12B). Note that the methods of using aclickable triazabutadiene according to TBD-6 for click chemistrycomprises removing of the protection group (e.g., deprotecting the silylgroup). In some embodiments, the use of a compound such as but notlimited to tetra-n-butylammonium fluoride (TBAF) in the reaction mayallow for deprotecting the silyl group to allow a click reaction toproceed.

The synthesis of TBD-7 (from FIG. 11B) was performed as shown in FIG.12C. Bis-mesitylimidazolium chloride salt Compound 16 was deprotonatedwith sodium hydride (NaH) to generate the reactive NHC species in thepresence of p-azido-methylbenzoate Compound 9 to form Compound 17. TheK-salt intermediate of Compound 7 underwent1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) coupling withhydroxybenzothiazole (HOBt) in order to form a reactive HOBt esterintermediate. This rendered the carbonyl group highly electrophilic fora nucleophilic attack by propargyl amine to produce the amide bond andthe triazabutadiene Compound 7 with a terminal alkyne for clickchemistry.

FIG. 12D shows click chemistry using a clickable triazabutadienecomprising an alkyne handle (TBD-6). Table 1 below shows examples of Rgroups attached to the azide handle that is clicked to the clickabletriazabutadiene. The triazabutadiene 6, organic azide, CuI, and PMEDTAwere in a solution of tetrahydrofuran (THF).

TABLE 1 Synthesis of imidazolium-substituted triazole-triazabutadieneEntry R Product Time (hr) Yield (%) 1

19 5 56 2

20 2 48

FIG. 12E shows click chemistry using a clickable triazabutadienecomprising an alkyne handle (TBD-7). Triazabutadiene Compound 7 (TBD-7)was subjected to similar conditions using the same azides (Table 2below) with a minor alteration. Due to the installation of the alkynehandle after triazabutadiene synthesis, a protection step wasunnecessary. The Cu-click reaction proceeded with moderate yields.

TABLE 2 Synthesis of aryl-substituted triazole-triazabutadient Entry RProduct Time (hr) Yield (%) 1

21 4 50 2

22 6 73

The present invention also features methods of cleaving saidtriazabutadienes, e.g., cleaving the clickable triazabutadienes that hasundergone click chemistry and is in the cross-linking state, e.g.,compounds such as the products of the reactions in FIG. 12D and FIG.12E. In some embodiments, cleavage of the cross-linking triazabutadieneliberates the diazonium species; thus, the present invention alsofeatures methods that feature diazonium reactions following cleavage ofsaid linking triazabutadienes.

The present invention also features a lysine-reactiveN-hydroxysuccinimide (NHS) modified triazabutadiene, e.g., Compound 68(see FIG. 13A). This was synthesized from bismestiyl imidazolium, e.g.,Compound 69 (FIG. 13B). A series of derivatives is shown in FIG. 13C.Several variants of Compound 68 may be synthesized and tested. Forexample, Compound 70 with a sulfonate-containing NHS ester may provide aprotein modified identically to using Compound 8, but it is may besoluble at higher concentrations, which may enable more rapid labelingof dilute protein samples (such as viral samples). Anothersulfonate-containing derivative, Compound 71, may have the effect ofadding a negative charge to the surface of the protein that it modifies.Because this is a lysine reactive probe, the change of the chargedsurface of the protein from positive to negative could have significantimpacts. That said, once the triazabutadiene degrades to an aryldiazonium ion, the surface will regain its positively charged nature. Athird probe that may be synthesized, Compound 72, contains a cysteine(thiol) reactive maleimide, which may offer a greater degree ofselectivity due to the low abundance of surface exposed thiols. Thisprobe may be used in conjunction with proteins that have been mutated topossess a solvent expose cysteine at positions of interest. Based onearlier studies that focused on the electronic-parameters associatedwith aryl diazonium release rates, the linkage chemistry to the arylring may have an effect. The N-linked amide probe, Compound 73, may beused to look at those electronic effects in the context of a complexbiological sample.

To assess the role of charges and perturbations that the probes have onproteins alkyne-containing triazabutadiene Compound 74 may besynthesized (see FIG. 14A). The alkyne may allow further modification ofthe scaffold either pre or post protein modification. The proof ofconcept version of this probe was shown to undergo a Cu(I) catalyzedclick reaction with p-azidotrifluoromethylbenzene (FIG. 14B).

The azide version, Compound 76, may help remove the limits on couplingpartners (FIG. 14C). To further expand coupling capabilities, an azideor alkyne on the aryl side of the triazabutadiene, like in Compound 78(FIG. 14D), may allow for conjugation to alkyne- or azide-modified(respectively) unnatural amino acids, glycans, or other metabolites thatare to be studied.

In the absence of a protein cross-linking event, there may be an aryldiazonium, which decomposes to a phenol and remains bound to the lysine.This phenol is likely prone to redox chemistry and as such represents anavenue for complexity during proteomic analysis. A self-immolatingtriazabutadiene has been designed to circumvent these pitfalls.Referring to FIG. 15, triazabutadiene Compound 80 provides an aryldiazonium (Compound 81), which can go on to react with locally availabletyrosine residues, or decompose to phenol Compound 82 if none areavailable. This phenol will further degrade via quinone-methidechemistry to extrude carbon dioxide and return an unaltered lysineresidue.

d. Diazonium Degradation for Cargo or Drug Release

In some embodiments, the triazabutadiene molecules of the presentinvention may be used in applications involving diazonium degradation torelease cargo or drugs. For example, a group of applications takesadvantage of the solvolysis of diazonium salts to produce phenolicbyproducts. The degradation of diazonium salts to phenols, via arylcations, is a first-order process that is not pH dependent in thephysiological range of pHs. The half-life of this first order processdepends on substitution on the aryl ring; the rate for benzenediazoniumis ˜4 hours. Indeed, the product of this degradation and subsequentazo-dye formation was observed if resorcinol is not put into thebuffered NMR experiments.

In some embodiments, the acid-dependent instability of thetriazabutadiene molecule may allow for a drug or cargo molecule to bedeposited at a desired location and time (e.g., the reaction can becontrolled and initiated at a desired time and location). As such, thepresent invention also features methods of delivering a drug (or a cargocompound) to a subject. In some embodiments, the method comprisesproviding a triazabutadiene molecule according to the present invention,conjugating a drug (or cargo compound) to the triazabutadiene molecule;and administering the conjugate (the drug/cargo-triazabutadieneconjugate) to the subject. In some embodiments, the method comprisesproviding a triazabutadiene molecule according to the present inventionwherein the triazabutadiene molecule comprises the drug (or cargocompound); and administering the triazabutadiene molecule to thesubject. In some embodiments, the diazonium species of thetriazabutadiene molecule is part of the drug (or cargo compound). Insome embodiments, the drug (or cargo compound) is formed when thediazonium species reacts to a phenol species. In some embodiments, thedrug is an anti-cancer drug. The drug (or cargo compound) is not limitedto an anti-cancer drug. Any appropriate drug for any appropriatecondition may be considered. Likewise, the triazabutadiene molecules maybe incorporated into drug/cargo-delivery systems for conditionsincluding but not limited to cancer or other conditions associated withlow pH states (e.g., gastrointestinal conditions, sepsis, ketoacidosis,etc.). Non-limiting examples of drugs (e.g., drugs that have a phenolicfunctional group, which may be masked as prodrugs) include: Abarelix,Alvimopan, Amoxicillin, Acetaminophen, Arformoterol, Cefadroxil,Cefpiramide, Cefprozil, Clomocycline, Daunorubicin, Dezocine,Epinephrine, Cetrolrelix, Etoposide, Crofelemer, Ezetimibe, Idarubicin,Ivacaftor, Hexachlorophene, Labetalol, Lanreotide, Levodopa,Caspofungin, Butorphanol, Buprenorphine, Dextrothyroxine, Doxorubicin,Dopamine, Dobutamine, Demeclocycline, Diflunisal, Dienestrol,Diethylstilbestrol, Doxycycline, Entacapone, Arbutamine, Apomorphine,Balsalazide, Capsaicin, Epirubicin, Esterified Estrogens, EstradiolValerate, Estrone, Estradiol, Ethinyl Estradiol, Fulvestrant, Goserelin,Fluorescein, Indacaterol, Levosalbutamol, Levothyroxine, Liothyronine,Lymecycline, Mitoxantrone, Monobenzone, Morphine, Masoprocol,Mycophenolic Acid, Phenylephrine, Phentolamine, Oxytetracycline,Rifaximin, Rifapentine, Oxymetazoline, Raloxifene, Tolcapone,Terbutaline, Tetracycline, Mesalamine, Metaraminol, Methyldopa,Minocycline, Nabilone, Nalbuphine, Nelfinavir, Propofol, Rotigotine,Ritodrine, Salbutamol, Sulfasalazine, Salmeterol, Tapentadol,Tigecycline, Tolterodine, Teniposide, Telavancin, Topotecan,Triptorelin, Tubacurarine, Valrubicin, Vancomycin, etc.

In some embodiments, drug delivery systems featuring triazabutadienemolecules may be enhanced with other reactions, e.g., enzymaticreactions. Such additional reactions may help provide appropriatespecificity of the drug delivery system or appropriate timing to thedrug delivery system.

Referring to FIG. 16, the triazabutadiene molecules of the presentinvention may be used for applications involving benzoquinone methides,e.g., it may be possible to synthesize derivatives that can undergoelimination via para-quinone methide chemistry (see FIG. 16A). Referringto FIG. 16A, after acidification, triazabutadiene Compound 41 maydecompose to diazonium salt (Compound 42). This reactive species maydecompose to a phenol (Compound 43), which itself decomposes to aquinone methide and may liberate the cargo molecule (Compound 44). Itmay be possible to modify the electronic properties of the central ringin order to influence the rates at each step. This system is may beuseful for these modifications because none of them are expected toaffect the cargo. The azide-coupling chemistry may render this amenableto wide variety of chemical cargos. In a biological context thesecompounds may be able to release their desired cargo upon entry into theendosome, or upon exposure to non-virally relevant acidic environmentssuch as in proximity to cancerous tumors. This type of attachmentchemistry may be utilized as a method for drug or detection delivery,and may have an added level of specificity if the system was deliveredto a desired location using an antibody or aptamer.

In some embodiments, Z¹ (see FIG. 1, FIG. 16C) is a prodrug comprising aphenolic functional group, wherein the phenolic group is masked as atriazylidene moiety. An example of how a prodrug is released (e.g., inan acidic environment, e.g., in a patient) is illustrated in FIG. 16B.Without wishing to limit the present invention to any theory ormechanism, it is believed that all drugs, such as those approved by theU.S. Food and Drug Administration, that have a phenolic functional groupmay be masked as a triazylidene moiety.

Referring to FIG. 16B, Compound C is a stimulant that is produced byGlaxo Smith-Kline Beecham pharmaceutical company. The phenolic group ofCompound C can be converted to an azide group, e.g., by displacement ofthe hydroxyl group with an azide group. In some embodiments, thephenolic group is first converted to a suitable leaving group beforesubjecting to a nucleophilic displacement reaction with an azide group.The resulting azide Compound A is then reacted with3-(3-mesityl-2-(phenyltriaz-2-en-1-ylidene)-2,3-dihydro-1H-imidazol-1-yl)propane-1-sulfonate to produce triazylidene Compound B. In someembodiments, when Compound B is administered to a patient (e.g., orallyor intravenously), the acidic environment of the patient'sgastrointestinal tract (if administered orally) or patient's bloodplasma (when administered intravenously) decomposes it to generate acorresponding diazonium compound regenerates the phenolic group asillustrated in FIG. 16B. By converting the phenolic group (e.g., thehydroxyl group that is attached to a phenyl ring) to an azide, oneskilled in the art having read the present application can readilyconvert the phenol compound to a triazylidene compound of the invention.Thus, the triazylidene moiety serves as a masking group for a phenolicfunctional group.

The present invention also features a method for administering a drugcomprising a phenolic function group to a subject in need of such a drugadministration. In some embodiments, the method comprises converting adrug comprising a phenolic-functional group to a prodrug, wherein saidprodrug comprises an acid labile triazylidene moiety; and administeringsaid prodrug to a subject in need of such a drug administration. In someembodiments, the triazylidene compound may also comprise a watersolubility conferring moiety and/or Y¹ functional group.

The present invention also features a method of converting a drugcomprising a phenolic-function group to an acid labile prodrug. In someembodiments, the phenolic-functional group is converted to an azidegroup. The azide functional group may then be reacted with a carbene toproduce an acid labile prodrug comprising a triazylidene moiety (seeFIG. 16D).

In some embodiments, a triazabutadiene molecule is conjugated to anothermolecule (a conjugate molecule), e.g., a protein (e.g., an amino acidsuch as but not limited to lysine), a lipid, or other appropriatemolecule. In some embodiments, the diazonium species part of thetriazabutadiene molecule is conjugated to the conjugate molecule. Insome embodiments, the cyclic guanidine species part of thetriazabutadiene molecule is conjugated to the conjugate molecule. Insome embodiments, the triazabutadiene molecule is attached to theconjugate molecule via a linker. Linkers are well known to one ofordinary skill in the art and may include (but are not limited to) apolyether linkers such as polyethylene glycol linkers. In someembodiments, the conjugate molecule to which the triazabutadienemolecule is conjugated comprises an antibody or a fragment thereof. Insome embodiments, the conjugate molecule to which the triazabutadienemolecule is conjugated comprises a viral protein.

In some embodiments, the triazabutadiene molecules of the presentinvention are used for pull-down studies wherein a biomolecule orprotein of interest is attached to one side and the other side isappended to something such as but not limited to a small molecule (e.g.,hapten such as biotin) or compound. Using biotin as an example, thebiomolecule or protein of interest can be pulled down using an avidinbead (which binds strongly to the biotin) and thoroughly washed. Thismay be useful for protein enrichment. The biomolecule or protein ofinterest may then be cleaved from the avidin bead by means of reductivecleavage of the triazabutadiene that holds them together. The presentinvention is not limited to these components, for example thisapplication could also feature the use of a probe (e.g., fluorescent orotherwise) attached to an antibody used to interrogate a complex sample.

In some embodiments, reductive cleavage of triazabutadiene molecules mayalso be used to cleave unreacted triazabutadienes that did not undergodiazonium formation/reaction chemistry that is associated with a drop inpH (or other mechanism) as described above (a sort of quench for the pHchemistry).

As previously discussed, the diazonium species can react with a phenolspecies such as resorcinol or other appropriate phenol species. In someembodiments, a phenol species or resorcinol species is conjugated to aprotein, e.g., a protein different from the protein to which thetriazabutadiene molecule is conjugated, a protein that is the sameprotein to which the triazabutadiene molecule is conjugated, etc. Insome embodiments, the resorcinol species or phenol species that thediazonium species reacts with is the phenol functional group of atyrosine residue.

c. Other Applications

As previously discussed, the present invention features triazabutadienesas adhesives. FIG. 17 shows a triazabutadiene molecule bonded to a firstsurface. A phenol-containing compound is bonded to a second surface.First and/or second surfaces may include but are not limited to glass,plastic, a biomaterial, or any other appropriate surface, e.g., asurface that allows for linkage chemistry, e.g., the first surface couldbe any surface that allows for the attachment of a triazabutadienemolecule, the second surface could be any surface that allows for theattachment of a phenol-containing compound. Non-limiting examples ofmaterials also include Tufnol materials such as phenolic cottonlaminated plastics, phenolic paper laminated plastics, etc., a phenolformaldehyde resin such as bakelite (or baekelite), etc. As in FIG. 17,the first reaction (wherein the triazabutadiene molecule is exposed towater to result in diazonium species formation) may be performed at roomtemperature; however, the reaction may be at a different temperature,e.g., depending on the environmental conditions. Without wishing tolimit the present invention to any theory or mechanism, it is believedthat different temperatures may affect the rate at which the firstreaction (wherein the triazabutadiene molecule is exposed to water toresult in diazonium species formation) and/or the second reaction(wherein the diazonium species reacts with the phenol-containingcompound on the second surface) occurs. FIG. 17 also shows cleavage ofthe azobenzene linkage upon treatment with the reducing agent sodiumdithionite. In some embodiments, the reducing agent is not sodiumdithionite but is another appropriate reducing agent. In someembodiments, the surface (e.g., glass, plastic, etc.) is modified, e.g.,using an etching mechanism. In some embodiments, photolithographyetching may be used to shape the available triazabutadiene molecules.For example, one may intentionally expose certain triazabutadienemolecules to light (e.g., in a pattern via a mask, for example) so as totransition them to the diazonium species; if left unreacted, thediazonium species will then transition to a phenolic compound (aspreviously described), and thus will be non-sticky or unreactive withthe phenol-containing compound on a second surface. This system canallow for the etching away of undesired triazabutadienes.

As previously discussed, the present invention features triazabutadienesas additives in adhesive systems. In some embodiments, triazabutadienesare used with (e.g., added to) adhesives systems such as existingadhesive systems (e.g., epoxy adhesive systems). Epoxy adhesive systemstypically comprise an epoxy compound (epoxy resin) and a co-reactant(curing agent or hardener), wherein the adhesive is formed when theco-reactant reacts with the epoxy compound. The present inventionfeatures formulations comprising a triazabutadiene and an epoxycompound, wherein the formulation is adapted to react with a curingagent (co-reactant) to form an adhesive. Epoxy resins and curing agentsare well known to one of ordinary skill in the art. Examples of epoxyresins include but are not limited to bisphenol A epoxy resins andglycidylamine epoxy resins. Examples of curing agents include but arenot limited to amines and thiols. Note that the triazabutadiene can beattached to either the amine or epoxy side. Similarly, the electron-richaryl (e.g., phenol) can similarly be added to either component (orboth).

In some embodiments, the triazabutadiene comprises an epoxide (e.g.,epoxide or other appropriate epoxy group). A non-limiting example of atriazabutadiene comprising an epoxide is shown as Compound 1 in FIG.18A. The epoxide triazabutadiene may be mixed with an epoxy residue(Compound B in FIG. 18A), e.g., to generate a formulation. Note that thepresent invention is not limited to the epoxy residue shown in FIG. 18A.In some embodiments, the formulation (formulation comprising thetriazabutadiene and the epoxy resin) further comprises an electron-richaryl ring group (e.g., phenol or other appropriate group) with anepoxide (see Compound 2). This may help provide additional electron richaryl rings (e.g., phenol groups) with which the aryl diazonium speciescan react (subsequent to subjecting the triazabutadiene to appropriateconditions so as to yield said aryl diazonium species). Also shown inFIG. 18A is a non-limiting example of a co-reactant (curing agent). Insome embodiments, an electron-rich aryl ring (e.g., phenol group) (seeCompound 2b) is added to the co-reactant. As previously discussed, thismay help provide additional electron rich aryl rings (e.g., phenolgroups) with which the aryl diazonium species can react.

The reaction of the formulation (Compound 1 and Compound B; or Compound1, Compound 2, and Compound B) and the co-reactant (Compound A; orCompound A and Compound 2b) yields Compound 3, e.g., a polymerizedtriazabutadiene. Exposure of Compound 3 to water (or other appropriateconditions such as acid) yields the aryl diazonium species (e.g.,Compound 4). Compound 4 is available for reacting with electron-richaryl rings, which can provide for the adhesive properties.

The present invention is not limited to triazabutadienes comprising anepoxide. For example, in some embodiments, the triazabutadiene anyappropriate class of polymer (e.g., for polymerization processes), e.g.,polystyrene, α-β-unsaturated ester acrylate, or the like. The class ofpolymer may be one that does not require heat for polymerization (ordoes not require heat such that the triazabutadiene functionalitieswould be compromised or destroyed). For reference, FIG. 18D showspolystyrene as if attached to the N₃ area of a triazabutadiene, e.g., inlieu of an epoxide as shown in Compound 1 of FIG. 18A. FIG. 18D alsoshows polyacrylamide as if attached to the N₃ area of a triazabutadiene,e.g., in lieu of an epoxide as shown in Compound 1 of FIG. 18A. In someembodiments, the triazabutadiene comprises an amine. In someembodiments, the triazabutadiene has a structure similar to that of FIG.18C, wherein the triazabutadiene is shortened relative to Compound 1 ofFIG. 18A (the epoxide is directly linked to the aryl ring). In someembodiments, the triazabutadiene is an azide-containing compound thatcan be clicked onto other compounds as desired.

The formulation may comprise any appropriate percentage oftriazabutadiene. For example, the formulation may comprise a particularpercentage of triazabutadiene that provides desired properties (e.g.,cure time, cure strength, color, melting/decomposition temperature,ability to heal (e.g., allow for initially unreacted triazabutadienemolecules to yield the diazonium species which subsequently bond tonearby phenol-containing compounds) of the adhesive or polymer.

In some embodiments, the formulation comprises from 0.01% to 0.1%triazabutadiene. In some embodiments, the formulation comprises from0.01% to 1% triazabutadiene. In some embodiments, the formulationcomprises from 0.01% to 10% triazabutadiene. In some embodiments, theformulation comprises from 0.01% to 20% triazabutadiene. In someembodiments, the formulation comprises from 0.1% to 1% triazabutadiene.In some embodiments, the formulation comprises from 0.1% to 10%triazabutadiene. In some embodiments, the formulation comprises from0.1% to 20% triazabutadiene. In some embodiments, the formulationcomprises from 0.1% to 30% triazabutadiene. In some embodiments, theformulation comprises from 0.1% to 40% triazabutadiene. In someembodiments, the formulation comprises from 0.1% to 50% triazabutadiene.In some embodiments, the formulation comprises from 1% to 10%triazabutadiene. In some embodiments, the formulation comprises from 1%to 20% triazabutadiene. In some embodiments, the formulation comprisesfrom 1% to 30% triazabutadiene. In some embodiments, the formulationcomprises from 1% to 40% triazabutadiene. In some embodiments, theformulation comprises from 1% to 50% triazabutadiene. In someembodiments, the formulation comprises from 1% to 60% triazabutadiene.In some embodiments, the formulation comprises from 1% to 70%triazabutadiene. In some embodiments, the formulation comprises from 1%to 80% triazabutadiene. In some embodiments, the formulation comprisesfrom 1% to 90% triazabutadiene. In some embodiments, the formulationcomprises between 10% to 20% triazabutadiene. In some embodiments, theformulation comprises between 20% to 30% triazabutadiene. In someembodiments, the formulation comprises between 30% to 40%triazabutadiene. In some embodiments, the formulation comprises between40% to more than 50% triazabutadiene.

In some embodiments, the formulation comprises about 0.01%triazabutadiene. In some embodiments, the formulation comprises about0.1% triazabutadiene. In some embodiments, the formulation comprisesabout 0.5% triazabutadiene. In some embodiments, the formulationcomprises about 1% triazabutadiene. In some embodiments, the formulationcomprises about 2% triazabutadiene. In some embodiments, the formulationcomprises about 5% triazabutadiene. In some embodiments, the formulationcomprises about 10% triazabutadiene. In some embodiments, theformulation comprises about 15% triazabutadiene. In some embodiments,the formulation comprises about 20% triazabutadiene. In someembodiments, the formulation comprises about 25% triazabutadiene. Insome embodiments, the formulation comprises about 30% triazabutadiene.In some embodiments, the formulation comprises about 40%triazabutadiene. In some embodiments, the formulation comprises about50% triazabutadiene. In some embodiments, the formulation comprises morethan about 50% triazabutadiene. The present invention is not limited tothe aforementioned percentages.

In some embodiments, the reaction of the triazabutadiene molecule to thediazonium species occurs in water within 10 seconds. In someembodiments, the reaction of the triazabutadiene molecule to thediazonium species occurs in water within 30 seconds. In someembodiments, the reaction of the triazabutadiene molecule to thediazonium species occurs in water within 1 minute. In some embodiments,the reaction of the triazabutadiene molecule to the diazonium speciesoccurs in water within 5 minutes. In some embodiments, the reaction ofthe triazabutadiene molecule to the diazonium species occurs in waterwithin 10 minutes. In some embodiments, the reaction of thetriazabutadiene molecule to the diazonium species occurs in water within15 minutes. In some embodiments, the reaction of the triazabutadienemolecule to the diazonium species occurs in water within 20 minutes. Insome embodiments, the reaction of the triazabutadiene molecule to thediazonium species occurs in water within 25 minutes. In someembodiments, the reaction of the triazabutadiene molecule to thediazonium species occurs in water within 30 minutes. In someembodiments, the reaction of the triazabutadiene molecule to thediazonium species occurs in water within 45 minutes. In someembodiments, the reaction of the triazabutadiene molecule to thediazonium species occurs in water within 60 minutes.

In some embodiments, the bonding of the diazonium species to thephenol-containing compound occurs within 10 seconds. In someembodiments, the bonding of the diazonium species to thephenol-containing compound occurs within 30 seconds. In someembodiments, the bonding of the diazonium species to thephenol-containing compound occurs within 1 minute. In some embodiments,the bonding of the diazonium species to the phenol-containing compoundoccurs within 5 minutes. In some embodiments, the bonding of thediazonium species to the phenol-containing compound occurs within 10minutes. In some embodiments, the bonding of the diazonium species tothe phenol-containing compound occurs within 15 minutes. In someembodiments, the bonding of the diazonium species to thephenol-containing compound occurs within 20 minutes. In someembodiments, the bonding of the diazonium species to thephenol-containing compound occurs within 25 minutes. In someembodiments, the bonding of the diazonium species to thephenol-containing compound occurs within 30 minutes. In someembodiments, the bonding of the diazonium species to thephenol-containing compound occurs within 45 minutes. In someembodiments, the bonding of the diazonium species to thephenol-containing compound occurs within 60 minutes.

In some embodiments, light can be used to speed up the reaction. In someembodiments, varying triazabutadienes amounts can be added to speed upor slow down the reaction. In some embodiments, a surplus oftriazabutadienes may be used, which may help allow for an amount oftriazabutadiene molecules that are unreacted (and those unreactedtriazabutadienes may be buried amongst other reacted compounds). Theseunreacted triazabutadienes that are buried may be useful in the event ofa break in the seal. For example, a break in the seal may cause water tothen react with the unreacted triazabutadiene molecules to yield thediazonium species, and those newly formed diazonium species can thensubsequently bond to nearby phenol-containing compounds to perhaps“heal” the break in the seal or strengthen the bond.

The present invention also features systems (or kits) comprising saidformulations, e.g., kits comprising a triazabutadiene (e.g.,triazabutadiene comprising an epoxide) and an epoxy resin. In someembodiments, the kit further comprises a co-reactant (or a formulationwith a co-reactant and an electron-rich aryl ring compound), wherein theformulation is adapted to react with the co-reactant to form anadhesive.

Triazabutadienes for use as additives to adhesive or polymerizationsystems may be synthesized in a variety of ways. FIG. 18B shows anon-limiting example of synthesis of a triazabutadiene (Compound 1 fromFIG. 18A) comprising an epoxide. For example, in some embodiments, arylazides that are appropriately functionalized (e.g., with an epoxide orfunctional group that can be converted to an alkyl azide) may be coupledwith N-heterocyclic carbenes to form the triazabutadiene core. Thepresent invention is not limited to the compound or step shown in FIG.18B. For example, in some embodiments, TBS-Cl (tert-butyldimethylsilylchloride) may be optional. In some embodiments, an alternative toepoxide is added to the triazabutadiene in lieu of epoxide.

As previously discussed, the covalent bond formed between thephenol-containing compound and the diazonium compound forms a coloredcompound. In some embodiments, the color is red, orange, or a mix of redand orange. In some embodiments, the formation of the color can be usedas a positive indicator that the bonding reaction has occurred.

As previously discussed, the diazonium species, if not reacted with thephenol-containing compound, can break down into a phenolic compound(e.g., the diazonium species will extrude nitrogen gas to generate anaryl cation that will rapidly be quenched by solvating water, thusgenerating the phenolic compound).

This reaction is typically much slower than the second reaction (whereinthe diazonium species reacts with the phenol-containing compound boundto the second surface). This phenomenon can allow for the unreacteddiazonium species to eventually become non-sticky, or unreactive, whichmay be beneficial in certain circumstances (e.g., photolithography).

Thus, without wishing to limit the present invention to any theory ormechanism, it is believed that the system and methods of the presentinvention are advantageous because the technology provides underwateradhesion, the adhesive bond may be colored (e.g., highly coloredazobenzene linkages), which may serve as a positive indicator that thedesired reaction has occurred; and/or the chemical compounds (e.g.,unreacted diazonium species) may degrade over time so that the unbondedsurface does not remain sticky (e.g., adapted for adhesion) permanently.

In some embodiments, triazabutadienes of the present invention are usedas after-market adhesives, e.g., formulations for application to anyappropriate surface. For example, the triazabutadienes may be coated onone side of a surface and then activated (e.g., with water) to activateadhesive properties.

In some embodiments, the triazabutadienes are used as or are used incombination with bio-adhesives (e.g., natural underwater adhesives suchas mussel adhesive proteins).

As previously discussed, the present invention features triazabutadienesthat can cross-react with existing chemistries, e.g., epoxy chemistry),e.g., an epoxide-containing compound, an amine containing compound, anazide-containing compound that can be clicked onto other compounds asrequired. As an example of synthesis, aryl azides that have beenappropriately functionalized (e.g., with an epoxide or functional groupthat can be converted to an alkyl azide) may be coupled withN-heterocyclic carbenes to form the triazabutadiene core. The presentinvention is not limited to this route.

As previously discussed, the properties of the formulations featuringthe triazabutadiene compounds (e.g., triazabutadiene compounds with theepoxy resins or the like) may be assessed. For example, in someembodiments, gel time/cure time is assessed (e.g., assessing if it islonger, shorter, or similar as compared to samples prepared in theabsence of the triazabutadiene additive). In some embodiments, curestrength is assessed (e.g., via break-strength). For example, small(e.g., 0.5×2×5 cm) molded ingots may be broken; strength may be comparedto samples prepared in the absence of the triazabutadiene additive. Insome embodiments, the color of the material (e.g., the final material)is assessed, e.g., color changes may be observed. In some embodiments,odor is assessed (e.g., is there a strong odor, is there a change inodor). In some embodiments, viscosity is assessed, e.g., as compared tosamples prepared in the absence of the triazabutadiene additive. In someembodiments, melting/decomposition temperature is assessed, e.g., viatesting in a melt-temp apparatus. In some embodiments, healing potentialis assessed, e.g., ability to enhance adhesive bonding (if broken) usingwater. For example, in some embodiments, ingots may be cracked andsubmerged into water (and broken faces pushed together) and then besubjected to break-strength test.

As previously discussed, the present invention features formulationscomprising a triazabutadiene molecule and an epoxide resin. In someembodiments, the epoxide resin comprises an aliphatic epoxide, e.g., amolecule according to compound B in FIG. 18A. In some embodiments,n=1-10. In some embodiments, n=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In someembodiments, n=greater than 10. In some embodiments, the epoxide resincomprises an electron rich aryl compound, e.g., a molecule according toCompound 2 of FIG. 18A. The present invention also features methods ofproducing adhesives. In some embodiments, the method comprises providinga composition A, e.g., Compound 1 of FIG. 18A and Compound B of FIG.18A; or Compound 1 of FIG. 18A and Compound B of FIG. 18A and Compound 2of FIG. 18A. The method may further comprise providing a composition B,e.g., Compound A of FIG. 18A; or Compound A of FIG. 18A and Compound 2bof FIG. 18A. In some embodiments, m=1-5. In some embodiments, m=1, 2, 3,4, or 5. In some embodiments, m is greater than 5. The method mayfurther comprise mixing composition A and composition B to form aproduct C, product C being the adhesives. In some embodiments, product Ccomprises Compound 3 of FIG. 18A.

Product C above is a non-limiting example of a product of Composition Aand Composition B. Product C is not limited to this structure. Forexample, in some embodiments, the epoxy is directly linked to the arylring. Note that the triazabutadiene can be attached to either the amineor epoxy side. In some embodiments, an amine is present on thetriazabutadiene (in which case it could be added into theepoxy-containing monomers). In some embodiments, the method furthercomprises exposing product C to water, whereby a diazonium species isformed from the triazabutadiene; the diazonium species can react with anelectron rich aryl compound (e.g., a phenol compound).

The present invention also features cross-linkers that respond toenvironmental triggers. This may allow for a chemical snapshot of a keymoment of an interaction.

Example 1—Synthesis of a Fluorescent Triazabutadiene Probe

Example 1 describes transformation of a triazabutadiene into afluorescent probe. The present invention is not limited to thecompositions and methods described herein.

Referring to FIG. 19, the fluorescent azide Compound 12 was coupled tothe scaffold Compound 7. The yield of triazabutadiene Compound 26 wasexcellent. This modification added a component to the triazabutadiene byrendering the diazonium portion (upon appropriate cleavage of thetriazabutadiene Compound 26) a fluorescent probe.

Example 2—Enhanced Functionalities of Triazabutadiene

Example 2 describes synthesis of a water-soluble triazabutadiene viaclick chemistry, a bi-functional triazabutadiene, and a triazabutadienecomprising an epoxide used to produce an alkyne handle. The presentinvention is not limited to the compositions and methods describedherein.

Referring to FIG. 20A, in some embodiments, triazabutadienes may be madewater soluble by attaching a water solubilizing agent or functionalgroup to the triazabutadiene via click chemistry as described herein.For example, FIG. 20A shows formation of a triazole with a tertiaryamine handle can undergo a nucleophilic attack on 1,3-propane sultone tosynthesize a water-soluble zwitterionic triazabutadiene.

FIG. 20B shows a bis-alkynyl triazabutadiene comprising two alkynehandles, one on the imidazole portion and one on the aryl portion (leftside). The right side of the figure shows the two alkyne handles clickedvia click chemistry with an azide group. A two-handled triazabutadienecan help enhance functionality of the triazabutadiene. For example, thetwo handles can be used to attach two different components (e.g.,biological components, e.g., a protein, a drug, etc.). For example, insome embodiments, one side is used for a first biological component andthe other side is used for a second biological component. In someembodiments, one side is used for a biological component and the otherside is used for a water-solubilizing component. The present inventionis not limited to the aforementioned attachment components or uses for atwo-handled triazabutadiene. In some embodiments, if one alkyne isprotected, the other side could be preferentially used for clicking.FIG. 21 shows a triazabutadiene comprising an epoxide (left) used toproduce a triazabutadiene with an alkyne group adapted for clickchemistry.

The disclosures of the following documents are incorporated in theirentirety by reference herein: U.S. Pat. No. 8,617,827; U.S. Pat.Application No. 2009/0048222; U.S. Pat. No. 3,591,575. U.S. Pat. No.3,607,542; U.S. Pat. No. 4,107,353; WO Pat. No. 2008090554; U.S. Pat.No. 4,218,279; U.S. Pat. App. No. 2009/0286308; U.S. Pat. No. 4,356,050;U.S. Pat. No. 8,603,451; U.S. Pat. No. 5,856,373; U.S. Pat. No.4,602,073; U.S. Pat. No. 3,959,210. The disclosures of the followingpublications are incorporated in their entirety by reference herein:Kimani and Jewett, 2015, Angewandte Chemie International Edition (DOI:10.1002/anie.201411277—Online ahead of print). Zhong et al., 2014,Nature Nanotechnology 9, 858-866; Stewart et al., 2011, J Polym Sci BPolym Phys 49(11):757-771; Poulsen et al., 2014, Biofouling30(4):513-23; Stewart, 2011, Appl Microbiol Biotechnol 89(1):27-33;Stewart et al., 2011, Adv Colloid Interface Sci 167(1-2):85-93;Hennebert et al., 2015, Interface Focus 5(1):2014.

Various modifications of the invention, in addition to those describedherein, will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims. Each reference cited in the presentapplication is incorporated herein by reference in its entirety.

Although there has been shown and described the preferred embodiment ofthe present invention, it will be readily apparent to those skilled inthe art that modifications may be made thereto which do not exceed thescope of the appended claims. Therefore, the scope of the invention isonly to be limited by the following claims. Reference numbers recited inthe claims are exemplary and for ease of review by the patent officeonly, and are not limiting in any way. In some embodiments, the figurespresented in this patent application are drawn to scale, including theangles, ratios of dimensions, etc. In some embodiments, the figures arerepresentative only and the claims are not limited by the dimensions ofthe figures. In some embodiments, descriptions of the inventionsdescribed herein using the phrase “comprising” includes embodiments thatcould be described as “consisting of”, and as such the writtendescription requirement for claiming one or more embodiments of thepresent invention using the phrase “consisting of” is met.

The reference numbers recited in the below claims are solely for ease ofexamination of this patent application, and are exemplary, and are notintended in any way to limit the scope of the claims to the particularfeatures having the corresponding reference numbers in the drawings.

What is claimed is:
 1. A clickable triazabutadiene according to FormulaC wherein both X¹ and X² comprise a terminal alkyne handle; wherein A=S,O, or N; D=H, —CH═CH—CH=E-, halides, cyano, sulfonates, alkyl chain, ortrifluoromethyl; E=H, —CH═CH—CH=D-, halides, cyano, sulfonates, alkylchain, or trifluoromethyl; and wherein Y¹ comprises a tri-substitutedaryl group; wherein the alkyne handle of X¹ is adapted to cross-link toan azide handle of a first linking component via click chemistry, andthe alkyne handle of X² is adapted to cross-link to an azide handle of asecond component via click chemistry, wherein the first linkingcomponent comprises a biological component and the second linkingcomponent comprises a functional group conferring water-solubility


2. The clickable triazabutadiene of claim 1, wherein the tri-substitutedaryl group of Y¹ comprises mesityl, a NHS-ester moiety, anoligonucleotide, a peptide; a fluorescence quencher; a pro-fluorophore;an alkyne; a triazene; an aldehyde; an amine; an aminooxy; a halogen; ora combination thereof.
 3. The clickable triazabutadiene of claim 1,wherein the biological component comprises a peptide, anoligonucleotide, or a drug.
 4. A clickable triazabutadiene according toFormula C wherein both X¹ and X² comprise a terminal alkyne handle;wherein A=S, O, or N; D=H, —CH═CH—CH=E-, halides, cyano, sulfonates,alkyl chain, or trifluoromethyl; E=H, —CH═CH—CH=D-, halides, cyano,sulfonates, alkyl chain, or trifluoromethyl; and wherein Y¹ comprises atri-substituted aryl group; wherein the alkyne handle of X¹ is adaptedto cross-link to an azide handle of a first linking component via clickchemistry, and the alkyne handle of X² is adapted to cross-link to anazide handle of a second component via click chemistry, wherein both thefirst linking component and the second linking component comprise abiological component


5. The clickable triazabutadiene of claim 4, wherein the tri-substitutedaryl group of Y¹ comprises mesityl, a NHS-ester moiety, anoligonucleotide, a peptide; a fluorescence quencher; a pro-fluorophore;an alkyne; a triazene; an aldehyde; an amine; an aminooxy; a halogen; ora combination thereof.
 6. The clickable triazabutadiene of claim 4,wherein the biological component comprises a peptide, anoligonucleotide, or a drug.
 7. A clickable triazabutadiene according toFormula C wherein both X¹ and X² comprise a terminal alkyne handle,wherein the alkyne handle of X¹ is adapted to cross-link to an azidehandle of a first linking component via click chemistry, and the alkynehandle of X² is adapted to cross-link to an azide handle of a secondcomponent via click chemistry


8. The clickable triazabutadiene of claim 7, wherein A=S, O, or N; D=H,—CH═CH—CH=E-, halides, cyano, sulfonates, alkyl chain, ortrifluoromethyl; E=H, —CH═CH—CH=D-, halides, cyano, sulfonates, alkylchain, or trifluoromethyl; and Y¹ comprises a tri-substituted arylgroup.
 9. The clickable triazabutadiene of claim 8, wherein thetri-substituted aryl group of Y¹ comprises mesityl, a NHS-ester moiety,an oligonucleotide, a peptide; a fluorescence quencher; apro-fluorophore; an alkyne; a triazene; an aldehyde; an amine; anaminooxy; a halogen; or a combination thereof.
 10. The clickabletriazabutadiene of claim 7, wherein the first linking componentcomprises a biological component and the second linking componentcomprises a functional group conferring water-solubility.
 11. Theclickable triazabutadiene of claim 10, wherein the biological componentcomprises a peptide, an oligonucleotide, or a drug.